Liquid Protein Formulations Containing Organophosphates

ABSTRACT

Concentrated, low-viscosity, low-volume liquid pharmaceutical formulations of proteins have been developed. Such formulations can be rapidly and conveniently administered by subcutaneous (SC) or intramuscular (IM) injection, rather than by lengthy intravenous infusion. These formulations include low-molecular-weight and/or high-molecular-weight proteins, such as mAbs, and organophosphates. The viscosity of the formulation is significantly reduced by the addition of one or more organophosphates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/890,044, filed on Feb. 6, 2018, which is a continuation of U.S.patent application Ser. No. 14/483,896, filed Sep. 11, 2014 (U.S. Pat.No. 9,913,905), which claims the benefit of U.S. Provisional ApplicationNo. 62/030,521, filed Jul. 29, 2014; U.S. Provisional Application No.62/026,497, filed Jul. 18, 2014; U.S. Provisional Application No.62/008,050, filed Jun. 5, 2014; U.S. Provisional Application No.61/988,005, filed May 2, 2014; U.S. Provisional Application No.61/946,436, filed Feb. 28, 2014; U.S. Provisional Application No.61/943,197, filed Feb. 21, 2014; U.S. Provisional Application No.61/940,227, filed Feb. 14, 2014; and U.S. Provisional Application No.61/876,621, filed Sep. 11, 2013. The disclosures of each of theseapplications are expressly incorporated herein by reference.

FIELD OF THE INVENTION

The invention is generally in the field of injectable pharmaceuticalformulations of proteins, such as monoclonal antibodies, and methods ofmaking and using thereof.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) are important protein-based therapeuticsfor treating various human diseases such as cancer, infectious diseases,inflammation, and autoimmune diseases. More than 20 mAb products havebeen approved by the U.S. Food and Drug Administration (FDA), andapproximately 20% of all biopharmaceuticals currently being evaluated inclinical trials are mAbs (Daugherty et al., Adv. Drug Deliv. Rev.58:686-706, 2006; and Buss et al., Curr. Opinion in Pharmacol.12:615-622, 2012).

mAb-based therapies are usually administered repeatedly over an extendedperiod of time and require several mg/kg dosing. Antibody solutions orsuspensions can be administered via parenteral routes, such as byintravenous (IV) infusions, and subcutaneous (SC) or intramuscular (IM)injections. The SC or IM routes reduce the treatment cost, increasepatient compliance, and improve convenience for patients and healthcareproviders during administration compared to the IV route. To beeffective and pharmaceutically acceptable, parenteral formulationsshould preferably be sterile, stable, injectable (e.g., via a syringe),and non-irritating at the site of injection, in compliance with FDAguidelines. Because of the small volumes required for subcutaneous(usually under about 2 mL) and intramuscular (usually under about 5 mL)injections, these routes of administration for high-dose proteintherapies require concentrated protein solutions. These highconcentrations often result in very viscous formulations that aredifficult to administer by injection, cause pain at the site ofinjection, are often imprecise, and/or may have decreased chemicaland/or physical stability.

These characteristics result in manufacturing, storage, and usagerequirements that can be challenging to achieve, in particular forformulations having high concentrations of high-molecular-weightproteins, such as mAbs. All protein therapeutics to some extent aresubject to physical and chemical instability, such as aggregation,denaturation, crosslinking, deamidation, isomerization, oxidation, andclipping (Wang et al., J. Pharm. Sci. 96:1-26, 2007). Thus, optimalformulation development is paramount in the development of commerciallyviable protein pharmaceuticals.

High protein concentrations pose challenges relating to the physical andchemical stability of the protein, as well as difficulty withmanufacture, storage, and delivery of the protein formulation. Oneproblem is the tendency of proteins to aggregate and form particulatesduring processing and/or storage, which makes manipulations duringfurther processing and/or delivery difficult. Concentration-dependentdegradation and/or aggregation are major challenges in developingprotein formulations at higher concentrations. In addition to thepotential for non-native protein aggregation and particulate formation,reversible self-association in aqueous solutions may occur, whichcontributes to, among other things, increased viscosity that complicatesdelivery by injection. (See, for example, Steven J. Shire et al., J.Pharm. Sci. 93:1390-1402, 2004.) Increased viscosity is one of the keychallenges encountered in concentrated protein compositions affectingboth production processes and the ability to readily deliver suchcompositions by conventional means. (See, for example, J. Jezek et al.,Advanced Drug Delivery Reviews 63:1107-1117, 2011.)

Highly viscous liquid formulations are difficult to manufacture, drawinto a syringe, and inject subcutaneously or intramuscularly. The use offorce in manipulating the viscous formulations can lead to excessivefrothing, which may further denature and inactivate the therapeuticallyactive protein. High viscosity solutions also require larger diameterneedles for injection and produce more pain at the injection site.

Currently available commercial mAb products administered by SC or IMinjection are usually formulated in aqueous buffers, such as a phosphateor L-histidine buffer, with excipients or surfactants, such as mannitol,sucrose, lactose, trehalose, POLOXAMER® (nonionic triblock copolymerscomposed of a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide))) or POLYSORBATE® 80(PEG(80)sorbitan monolaurate), to prevent aggregation and improvestability. Reported antibody concentrations formulated as describedabove are typically up to about 100 mg/mL (Wang et al., J. Pharm. Sci.96:1-26, 2007).

U.S. Pat. No. 7,758,860 describes reducing the viscosity in formulationsof low-molecular-weight proteins using a buffer and a viscosity-reducinginorganic salt, such as calcium chloride or magnesium chloride. Thesesame salts, however, showed little effect on the viscosity of ahigh-molecular-weight antibody (IMA-638) formulation. As described inU.S. Pat. No. 7,666,413, the viscosity of aqueous formulations ofhigh-molecular-weight proteins has been reduced by the addition of suchsalts as arginine hydrochloride, sodium thiocyanate, ammoniumthiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zincchloride, or sodium acetate in a concentration of greater than about 100mM or, as described in U.S. Pat. No. 7,740,842, by addition of organicor inorganic acids. However, these salts do not reduce the viscosity toa desired level and in some cases make the formulation so acidic that itis likely to cause pain at the site of injection.

U.S. Pat. No. 7,666,413 describes reduced-viscosity formulationscontaining specific salts and a reconstituted anti-IgE mAb, but with amaximum antibody concentration of only up to about 140 mg/mL. U.S. Pat.No. 7,740,842 describes E25 anti-IgE mAb formulations containingacetate/acetic acid buffer with antibody concentrations up to 257 mg/mL.The addition of salts such as NaCl, CaCl₂), or MgCl₂ was demonstrated todecrease the dynamic viscosity under high-shear conditions; however, atlow-shear the salts produced an undesirable and dramatic increase in thedynamic viscosity. Additionally, inorganic salts such as NaCl may lowersolution viscosity and/or decrease aggregation (EP 1981824).

Non-aqueous antibody or protein formulations have also been described.WO2006/071693 describes a non-aqueous suspension of up to 100 mg/mL mAbin a formulation having a viscosity enhancer (polyvinylpyrrolidone, PVP)and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes 100mg/mL non-aqueous lysozyme suspension formulations containing PVP,glycofurol, benzyl benzoate, benzyl alcohol, or PEG 400.US2008/0226689A1 describes 100 mg/mL human growth hormone (hGH) singlephase, three vehicle component (polymer, surfactant, and a solvent),non-aqueous, viscous formulations. U.S. Pat. No. 6,730,328 describesnon-aqueous, hydrophobic, non-polar vehicles of low reactivity, such asperfluorodecalin, for protein formulations. These formulations arenon-optimal and have high viscosities that impair processing,manufacturing and injection; lead to the presence of multiple vehiclecomponents in the formulations; and present potential regulatorychallenges associated with using polymers not yet approved by the FDA.

Alternative non-aqueous protein or antibody formulations have beendescribed using organic solvents, such as benzyl benzoate (Miller etal., Langmuir 26:1067-1074, 2010), benzyl acetate, ethanol, or methylethyl ketone (Srinivasan et al., Pharm. Res. 30:1749-1757, 2013). Inboth instances, viscosities of less than 50 centipoise (cP) wereachieved upon formulation at protein concentrations of at least about200 mg/mL. U.S. Pat. No. 6,252,055 describes mAb formulations withconcentrations ranging from 100 mg/mL up to 257 mg/mL. Formulations withconcentrations greater than about 189 mg/mL demonstrated dramaticallyincreased viscosities, low recovery rates, and difficulty in processing.U.S. Patent Application Publication No. 2012/0230982 describes antibodyformulations with concentrations of 100 mg/mL to 200 mg/mL. None ofthese formulations are low enough viscosity for ease of injection.

Du and Klibanov (Biotechnology and Bioengineering 108:632-636, 2011)described reduced viscosity of concentrated aqueous solutions of bovineserum albumin with a maximum concentration up to 400 mg/mL and bovinegamma globulin with a maximum concentration up to 300 mg/mL. Guo et al.(Pharmaceutical Research 29:3102-3109, 2012) described low-viscosityaqueous solutions of four model mAbs achieved using hydrophobic salts.The mAb formulation employed by Guo had an initial viscosity, prior toadding salts, no greater than 73 cP. The viscosities of manypharmaceutically important mAbs, on the other hand, can exceed 1,000 cPat therapeutically relevant concentrations.

It is not a trivial matter to control aggregation and viscosity inhigh-concentration mAb solutions (EP 2538973). This is evidenced by thefew mAb products currently on the market as high-concentrationformulations (>100 mg/mL) (EP 2538973).

The references cited above demonstrate that while many groups haveattempted to prepare low-viscosity formulations of mAbs and othertherapeutically important proteins, a truly useful formulation for manyproteins has not yet been achieved. Notably, many of the above reportsemploy agents for which safety and toxicity profiles have not been fullyestablished. These formulations would therefore face a higher regulatoryburden prior to approval than formulations containing compounds known tobe safe. Indeed, even if a compound were to be shown to substantiallyreduce viscosity, the compound may ultimately be unsuitable for use in aformulation intended for injection into a human.

Many pharmaceutically important high-molecular-weight proteins, such asmAbs, are currently administered via IV infusions in order to delivertherapeutically effective amounts of protein due to problems with highviscosity and other properties of concentrated solutions of largeproteins. For example, to provide a therapeutically effective amount ofmany high-molecular-weight proteins, such as mAbs, in volumes less thanabout 2 mL, protein concentrations greater than 150 mg/mL are oftenrequired.

It is, therefore, an object of the present invention to provideconcentrated, low-viscosity liquid formulations of pharmaceuticallyimportant proteins, especially high-molecular-weight proteins, such asmAbs.

It is a further object of the present invention to provide concentratedlow-viscosity liquid formulations of proteins, especiallyhigh-molecular-weight proteins, such as mAbs, capable of deliveringtherapeutically effective amounts of these proteins in volumes usefulfor SC and IM injections.

It is a further object of the present invention to provide theconcentrated liquid formulations of proteins, especiallyhigh-molecular-weight proteins, such as mAbs, with low viscosities thatcan improve injectability and/or patient compliance, convenience, andcomfort.

It is also an object of the present invention to provide methods formaking and storing concentrated, low-viscosity formulations of proteins,especially high-molecular-weight proteins, such as mAbs.

It is an additional object of the present invention to provide methodsof administering low-viscosity, concentrated liquid formulations ofproteins, especially high-molecular-weight proteins, such as mAbs. It isan additional object of the present invention to provide methods forprocessing reduced-viscosity, high-concentration biologics withconcentration and filtration techniques known to those skilled in theart.

SUMMARY OF THE INVENTION

Concentrated, low-viscosity, low-volume liquid pharmaceuticalformulations of proteins have been developed. Such formulations can berapidly and conveniently administered by subcutaneous (SC) orintramuscular (IM) injection, rather than by lengthy intravenousinfusion. These formulations include low-molecular-weight and/orhigh-molecular-weight proteins, such as mAbs, and organophosphates.Representative organophosphates include thiamine pyrophosphate (TPP),adenosine triphosphate (ATP), deoxyadenosine triphosphate (dATP),deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP),deoxycytidine triphosphate (dCTP), cyclic adenosine monophosphate(cAMP), nicotinamide adenine dinucleotide phosphate (NADP⁺), andpyridoxal phosphate, as well as salts thereof, at concentrationspreferably between about 0.01 M and about 0.50 M, most preferablybetween about 0.05 M and about 0.25 M.

The concentration of proteins is between about 10 mg/mL and about 5,000mg/mL, more preferably from about 100 mg/mL, up to about 2,000 mg/mL. Insome embodiments, the concentration of proteins is between about 100mg/mL to about 500 mg/mL, more preferably from about 300 mg/mL up toabout 500 mg/mL. Formulations containing proteins and organophosphatesare stable when stored at a temperature of 4° C., for a period of atleast one month, preferably at least two months, and most preferably atleast three months. The viscosity of the formulation is less than about75 cP, preferably below 50 cP, and most preferably below 20 cP at about25° C. In some embodiments, the viscosity is less than about 15 cP oreven less than or about 10 cP at about 25° C. In certain embodiments,the viscosity of the formulation is about 10 cP. Formulations containingproteins and organophosphates typically are measured at shear rates fromabout 0.6 s⁻¹ to about 450 s⁻¹, and preferably from about 2 s⁻¹ to about400 s⁻¹, when measured using a cone and plate viscometer. Formulationscontaining proteins and organophosphates typically are measured at shearrates from about 3 s⁻¹ to about 55,000 s⁻¹, and preferably from about 20s⁻¹ to about 2,000 s⁻¹ when measured using a microfluidic viscometer.

The viscosity of the protein formulation is reduced by the presence ofone or more viscosity-reducing organophosphates. Unless specificallystated otherwise, the term “viscosity-reducing organophosphate” includesboth single compounds and mixtures of two or more compounds. It ispreferred that the viscosity-reducing organophosphate(s) is (are)present in the formulation at a concentration less than about 1.0 M,preferably less than about 0.50 M, more preferably less than about 0.30M, and most preferably less than about 0.15 M. The formulations can havea viscosity that is at least about 30% less, preferably at least about50% less, most preferably at least about 75% less, than the viscosity ofthe corresponding formulation under the same conditions except forreplacement of the viscosity-reducing organophosphate with anappropriate buffer or salt of about the same concentration. In someembodiments, a low-viscosity formulation is provided where the viscosityof the corresponding formulation without the viscosity-reducingorganophosphate is greater than about 200 cP, greater than about 500 cP,or even above about 1,000 cP. In preferred embodiment, the shear rate ofthe formulation is at least about 0.5 s⁻¹, when measured using a coneand plate viscometer or at least about 1.0 s⁻¹, when measured using amicrofluidic viscometer.

For embodiments in which the protein is a “high-molecular-weightprotein”, the high molecular weight protein may have a molecular weightbetween about 100 kDa and about 500 kDa, preferably between about 120kDa and about 1,000 kDa, and most preferably between about 120 kDa andabout 250 kDa. The high-molecular-weight protein can be an antibody,such as a mAb, or a PEGylated or otherwise a derivatized form thereof.Preferred mAbs include natalizumab (TYSABRI®), cetuximab (ERBITUX®),bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab(REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab(ARZERRA®), and biosimilars thereof. The high-molecular-weight protein,optionally PEGylated, can be an enzyme. Other proteins and mixtures ofproteins may also be formulated to reduce their viscosity.

In some embodiments, the protein and viscosity-reducing organophosphateare provided in a lyophilized dosage unit, sized for reconstitution witha sterile aqueous pharmaceutically acceptable vehicle, to yield theconcentrated low-viscosity liquid formulations. The presence of theviscosity-reducing organophosphate(s) facilitates and/or accelerates thereconstitution of the lyophilized dosage unit compared to a lyophilizeddosage unit not containing a viscosity-reducing organophosphate.

Methods are provided herein for preparing concentrated, low-viscosityliquid formulations of high-molecular-weight proteins such as mAbs, aswell as methods for storing the low-viscosity, high-concentrationprotein formulations, and for administration thereof to patients. Inanother embodiment, the viscosity-reducing organophosphate is added tofacilitate processing (e.g., pumping, concentration, and/or filtration)by reducing the viscosity of the protein solutions. The concentration ofthe high-molecular-weight protein herein is between about 10 mg/mL andabout 5,000 mg/mL, most preferably from about 100 mg/mL up to about2,000 mg/mL. The viscosity of the formulation is less than about 75 cP,preferably below 50 cP, and most preferably below 20 cP. In someembodiments, the viscosity is less than about 15 cP or even less thanabout 10 cP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the viscosity (cP) of aqueous solutions of biosimilarAVASTIN® as a function of protein concentration (mg/mL) with 0.25 Mphosphate buffer, 0.10 M thiamine pyrophosphate (TPP), or 0.10 MTPP1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “protein,” as generally used herein, refers to a polymer ofamino acids linked to each other by peptide bonds to form a polypeptidefor which the chain length is sufficient to produce at least adetectable tertiary structure. Proteins having a molecular weight(expressed in kDa wherein “Da” stands for “Daltons” and 1 kDa=1,000 Da)greater than about 100 kDa may be designated “high-molecular-weightproteins,” whereas proteins having a molecular weight less than about100 kDa may be designated “low-molecular-weight proteins.” The term“low-molecular-weight protein” excludes small peptides lacking therequisite of at least tertiary structure necessary to be considered aprotein. Protein molecular weight may be determined using standardmethods known to one skilled in the art, including, but not limited to,mass spectrometry (e.g., ESI, MALDI) or calculation from known aminoacid sequences and glycosylation. Proteins can be naturally occurring ornon-naturally occurring, synthetic, or semi-synthetic.

“Essentially pure protein(s)” and “substantially pure protein(s)” areused interchangeably herein and refer to a composition comprising atleast about 90% by weight pure protein, preferably at least about 95%pure protein by weight. “Essentially homogeneous” and “substantiallyhomogeneous” are used interchangeably herein and refer to a compositionwherein at least about 90% by weight of the protein present is acombination of the monomer and reversible di- and oligo-meric associates(not irreversible aggregates), preferably at least about 95%.

The term “antibody,” as generally used herein, broadly covers mAbs(including full-length antibodies which have an immunoglobulin Fcregion), antibody compositions with polyepitopic specificity, bispecificantibodies, diabodies, and single-chain antibody molecules, as well asantibody fragments (e.g., Fab, Fab′, F(ab′)2, and Fv), single domainantibodies, multivalent single domain antibodies, Fab fusion proteins,and fusions thereof.

The term “monoclonal antibody” or “mAb,” as generally used herein,refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical, except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single epitope. These aretypically synthesized by culturing hybridoma cells, as described byKohler et al. (Nature 256: 495, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), or isolated from phageantibody libraries using the techniques described in Clackson et al.(Nature 352: 624-628, 1991) and Marks et al. (J. Mol. Biol. 222:581-597, 1991), for example. As used herein, “mAbs” specifically includederivatized antibodies, antibody-drug conjugates, and “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is (are)identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

An “antibody fragment” comprises a portion of an intact antibody,including the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870;Zapata et al., Protein Eng. 8:1057-1062, 1995); single-chain antibodymolecules; multivalent single domain antibodies; and multispecificantibodies formed from antibody fragments.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences ofantibodies) of mostly human sequences, which contain minimal sequencesderived from non-human immunoglobulin. (See, e.g., Jones et al., Nature321:522-525, 1986; Reichmann et al., Nature 332:323-329, 1988; andPresta, Curr. Op. Struct. Biol. 2:593-596, 1992.)

“Rheology” refers to the study of the deformation and flow of matter.

“Viscosity” refers to the resistance of a substance (typically a liquid)to flow. Viscosity is related to the concept of shear force; it can beunderstood as the effect of different layers of the fluid exertingshearing force on each other, or on other surfaces, as they move againsteach other. There are several measures of viscosity. The units ofviscosity are Ns/m², known as Pascal-seconds (Pa-s). Viscosity can be“kinematic” or “absolute”. Kinematic viscosity is a measure of the rateat which momentum is transferred through a fluid. It is measured inStokes (St). The kinematic viscosity is a measure of the resistive flowof a fluid under the influence of gravity. When two fluids of equalvolume and differing viscosity are placed in identical capillaryviscometers and allowed to flow by gravity, the more viscous fluid takeslonger than the less viscous fluid to flow through the capillary. If,for example, one fluid takes 200 seconds (s) to complete its flow andanother fluid takes 400 s, the second fluid is called twice as viscousas the first on a kinematic viscosity scale. The dimension of kinematicviscosity is length²/time. Commonly, kinematic viscosity is expressed incentiStokes (cSt). The SI unit of kinematic viscosity is mm²/s, which isequal to 1 cSt. The “absolute viscosity,” sometimes called “dynamicviscosity” or “simple viscosity,” is the product of kinematic viscosityand fluid density. Absolute viscosity is expressed in units ofcentipoise (cP). The SI unit of absolute viscosity is themilliPascal-second (mPa-s), where 1 cP=1 mPa-s. Viscosity may bemeasured by using, for example, a viscometer at a given shear rate ormultiple shear rates. An “extrapolated zero-shear” viscosity can bedetermined by creating a best fit line of the four highest-shear pointson a plot of absolute viscosity versus shear rate, and linearlyextrapolating viscosity back to zero-shear. Alternatively, for aNewtonian fluid, viscosity can be determined by averaging viscosityvalues at multiple shear rates. Viscosity can also be measured using amicrofluidic viscometer at single or multiple shear rates (also calledflow rates), wherein absolute viscosity is derived from a change inpressure as a liquid flows through a channel. Viscosity equals shearstress over shear rate. Viscosities measured with microfluidicviscometers can, in some embodiments, be directly compared toextrapolated zero-shear viscosities, for example those extrapolated fromviscosities measured at multiple shear rates using a cone and plateviscometer.

“Shear rate” refers to the rate of change of velocity at which one layerof fluid passes over an adjacent layer. The velocity gradient is therate of change of velocity with distance from the plates. This simplecase shows the uniform velocity gradient with shear rate (v¹−v₂)/h inunits of (cm/sec)/(cm)=1/sec. Hence, shear rate units are reciprocalseconds or, in general, reciprocal time. For a microfluidic viscometer,change in pressure and flow rate are related to shear rate. “Shearrate”, is used to refer to the speed with which a material is deformed.Formulations containing proteins and organophosphates are typicallymeasured at shear rates ranging from about 0.5 s⁻¹ to about 200 s⁻¹ whenmeasured using a cone and plate viscometer and a spindle appropriatelychosen by one skilled in the art to accurately measure viscosities inthe viscosity range of the sample of interest (i.e., a sample of 20 cPis most accurately measured on a CPE40 spindle affixed to a DV2Tviscometer (Brookfield)); greater than about 20 s⁻¹ to about 3,000 s⁻¹when measured using a microfluidic viscometer.

For classical “Newtonian” fluids, as generally used herein, viscosity isessentially independent of shear rate. For “non-Newtonian fluids,”however, viscosity either decreases or increases with increasing shearrate, e.g., the fluids are “shear thinning” or “shear thickening”,respectively. In the case of concentrated (i.e., high-concentration)protein solutions, this may manifest as pseudoplastic shear-thinningbehavior, i.e., a decrease in viscosity with shear rate.

The term “chemical stability,” as generally used herein, refers to theability of the protein components in a formulation to resist degradationvia chemical pathways, such as oxidation, deamidation, or hydrolysis. Aprotein formulation is typically considered chemically stable if lessthan about 5% of the components are degraded after 24 months at 4° C.

The term “physical stability,” as generally used herein, refers to theability of a protein formulation to resist physical deterioration, suchas aggregation. A formulation that is physically stable forms only anacceptable percentage of irreversible aggregates (e.g., dimers, trimers,or other aggregates) of the bioactive protein agent. The presence ofaggregates may be assessed in a number of ways, including by measuringthe average particle size of the proteins in the formulation by means ofdynamic light scattering. A formulation is considered physically stableif less than about 5% irreversible aggregates are formed after 24 monthsat 4° C. Acceptable levels of aggregated contaminants ideally would beless than about 2%. Levels as low as about 0.2% are achievable, althoughapproximately 1% is more typical.

The term “stable formulation,” as generally used herein, means that aformulation is both chemically stable and physically stable. A stableformulation may be one in which more than about 95% of the bioactiveprotein molecules retain bioactivity in a formulation after 24 months ofstorage at 4° C. or equivalent solution conditions at an elevatedtemperature, such as one month storage at 40° C. Various analyticaltechniques for measuring protein stability are available in the art andare reviewed, for example, in Peptide and Protein Drug Delivery,247-301, Vincent Lee, Ed., Marcel Dekker, Inc., New York, N.Y. (1991)and Jones, A., Adv. Drug Delivery Revs. 10:29-90, 1993. Stability can bemeasured at a selected temperature for a certain time period. For rapidscreening, for example, the formulation may be kept at 40° C., for 2weeks to one month, at which time residual biological activity ismeasured and compared to the initial condition to assess stability. Whenthe formulation is to be stored at 2° C.-8° C., generally theformulation should be stable at 30° C. or 40° C. for at least one monthand/or stable at 2° C.-8° C. for at least 2 years. When the formulationis to be stored at room temperature, about 25° C., generally theformulation should be stable for at least 2 years at about 25° C. and/orstable at 40° C. for at least about 6 months. The extent of aggregationfollowing lyophilization and storage can be used as an indicator ofprotein stability. In some embodiments, the stability is assessed bymeasuring the particle size of the proteins in the formulation. In someembodiments, stability may be assessed by measuring the activity of aformulation using standard biological activity or binding assays wellwithin the abilities of one ordinarily skilled in the art.

The term protein “particle size,” as generally used herein, means theaverage diameter of the predominant population of bioactive moleculeparticulates, or particle size distributions thereof, in a formulationas determined by using well known particle sizing instruments, forexample, dynamic light scattering, SEC (size exclusion chromatography),or other methods known to one ordinarily skilled in the art.

The term “concentrated” or “high-concentration”, as generally usedherein, describes liquid formulations having a final concentration ofprotein greater than about 10 mg/mL, preferably greater than about 50mg/mL, more preferably greater than about 100 mg/mL, still morepreferably greater than about 200 mg/mL, or most preferably greater thanabout 250 mg/mL.

A “reconstituted formulation,” as generally used herein, refers to aformulation which has been prepared by dissolving a dry powder,lyophilized, spray-dried or solvent-precipitated protein in a diluent,such that the protein is dissolved or dispersed in aqueous solution foradministration.

A “lyoprotectant” is a substance which, when combined with a protein,significantly reduces chemical and/or physical instability of theprotein upon lyophilization and/or subsequent storage. Exemplarylyoprotectants include sugars and their corresponding sugar alcohols,such as sucrose, lactose, trehalose, dextran, erythritol, arabitol,xylitol, sorbitol, and mannitol; amino acids, such as arginine orhistidine; lyotropic salts, such as magnesium sulfate; polyols, such aspropylene glycol, glycerol, poly(ethylene glycol), or poly(propyleneglycol); and combinations thereof. Additional exemplary lyoprotectantsinclude gelatin, dextrins, modified starch, and carboxymethyl cellulose.Preferred sugar alcohols are those compounds obtained by reduction ofmono- and di-saccharides, such as lactose, trehalose, maltose,lactulose, and maltulose. Additional examples of sugar alcohols areglucitol, maltitol, lactitol and isomaltulose. The lyoprotectant isgenerally added to the pre-lyophilized formulation in a “lyoprotectingamount.” This means that, following lyophilization of the protein in thepresence of the lyoprotecting amount of the lyoprotectant, the proteinessentially retains its physical and chemical stability and integrity.

A “diluent” or “carrier,” as generally used herein, is apharmaceutically acceptable (i.e., safe and non-toxic for administrationto a human or another mammal) and useful ingredient for the preparationof a liquid formulation, such as an aqueous formulation reconstitutedafter lyophilization. Exemplary diluents include sterile water,bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution, and combinations thereof.

A “preservative” is a compound which can be added to the formulationsherein to reduce contamination by and/or action of bacteria, fungi, oranother infectious agent. The addition of a preservative may, forexample, facilitate the production of a multi-use (multiple-dose)formulation. Examples of potential preservatives includeoctadecyldimethylbenzylammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chained), and benzethoniumchloride. Other types of preservatives include aromatic alcohols such asphenol, butyl and benzyl alcohol, alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, andm-cresol.

A “bulking agent,” as generally used herein, is a compound which addsmass to a lyophilized mixture and contributes to the physical structureof the lyophilized cake (e.g. facilitates the production of anessentially uniform lyophilized cake which maintains an open porestructure). Exemplary bulking agents include mannitol, glycine, lactose,modified starch, poly(ethylene glycol), and sorbitol.

A “therapeutically effective amount” is the least concentration requiredto effect a measurable improvement or prevention of any symptom or aparticular condition or disorder, to effect a measurable enhancement oflife expectancy, or to generally improve patient quality of life. Thetherapeutically effective amount is dependent upon the specificbiologically active molecule and the specific condition or disorder tobe treated. Therapeutically effective amounts of many proteins, such asthe mAbs described herein, are well known in the art. Thetherapeutically effective amounts of proteins not yet established or fortreating specific disorders with known proteins, such as mAbs, to beclinically applied to treat additional disorders may be determined bystandard techniques which are well within the craft of a skilledartisan, such as a physician.

The term “injectability” or “syringeability,” as generally used herein,refers to the injection performance of a pharmaceutical formulationthrough a syringe equipped with an 18-32 gauge needle. Injectabilitydepends upon factors such as pressure or force required for injection,evenness of flow, aspiration qualities, and freedom from clogging.Injectability of the liquid pharmaceutical formulations may be assessedby comparing the injection force of a reduced-viscosity formulation to astandard formulation without added viscosity-reducing organophosphates.The reduction in the injection force of the formulation containing anviscosity-reducing organophosphate reflects improved injectability ofthat formulation. The reduced viscosity formulations have improvedinjectability when the injection force is reduced by at least 10%,preferably by at least 30%, more preferably by at least 50%, and mostpreferably by at least 75% when compared to a standard formulationhaving the same concentration of protein under otherwise the sameconditions, except for replacement of the viscosity-reducingorganophosphate with an appropriate buffer of about the sameconcentration. Alternatively, injectability of the liquid pharmaceuticalformulations may be assessed by comparing the time required to injectthe same volume, such as 0.5 mL, or more preferably about 1 mL, ofdifferent liquid protein formulations when the syringe is depressed withthe same force.

The term “injection force,” as generally used herein, refers to theforce required to push a given liquid formulation through a givensyringe equipped with a given needle gauge at a given injection speed.The injection force is typically reported in Newtons. For example, theinjection force may be measured as the force required to push a liquidformulation through a 1 mL plastic syringe having a 0.25 inch insidediameter, equipped with a 0.50 inch 27 gauge needle at a 250 mm/mininjection speed. Testing equipment can be used to measure the injectionforce. When measured under the same conditions, a formulation with lowerviscosity will generally require an overall lower injection force.

In one embodiment, the injection is administered with a 27 gauge needleand the injection force is less than 30 N. The formulations can beadministered in most cases using a very small gauge needle, for example,between 27 and 31 gauge, typically 27, 29 or 31 gauge, optionally thinwalled.

The “viscosity gradient,” as used herein, refers to the rate of changeof the viscosity of a protein solution as protein concentrationincreases. The viscosity gradient can be approximated from a plot of theviscosity as a function of the protein concentration for a series offormulations that are otherwise the same but have different proteinconcentrations. The viscosity increases approximately exponentially withincreasing protein concentration. The viscosity gradient at a specificprotein concentration can be approximated from the slope of a linetangent to the plot of viscosity as a function of protein concentration.The viscosity gradient can be approximated from a linear approximationto the plot of viscosity as a function of any protein concentration orover a narrow window of protein concentrations. In some embodiments aformulation is said to have a decreased viscosity gradient if, when theviscosity as a function of protein concentration is approximated as anexponential function, the exponent of the exponential function issmaller than the exponent obtained for the otherwise same formulationwithout the viscosity-reducing organophosphate. In a similar manner, aformulation can be said to have a lower/higher viscosity gradient whencompared to a second formulation if the exponent for the formulation islower/higher than the exponent for the second formulation. The viscositygradient can be numerically approximated from a plot of the viscosity asa function of protein concentration by other methods known to theskilled formulation researchers.

The term “reduced-viscosity formulation,” as generally used herein,refers to a liquid formulation having a high concentration of ahigh-molecular-weight protein, such as a mAb, or a low-molecular-weightprotein that is modified by the presence of one or more additives tolower the viscosity, as compared to a corresponding formulation thatdoes not contain the viscosity-lowering additive(s).

The term “osmolarity,” as generally used herein, refers to the totalnumber of dissolved components per liter. Osmolarity is similar tomolarity but includes the total number of moles of dissolved species insolution. An osmolarity of 1 Osm/L means there is 1 mole of dissolvedcomponents per L of solution. Some solutes, such as ionic solutes thatdissociate in solution, will contribute more than 1 mole of dissolvedcomponents per mole of solute in the solution. For example, NaCldissociates into Na⁺ and Cl⁻ in solution and thus provides 2 moles ofdissolved components per 1 mole of dissolved NaCl in solution.Physiological osmolarity is typically in the range of about 280 mOsm/Lto about 310 mOsm/L.

The term “tonicity,” as generally used herein, refers to the osmoticpressure gradient resulting from the separation of two solutions by asemi-permeable membrane. In particular, tonicity is used to describe theosmotic pressure created across a cell membrane when a cell is exposedto an external solution. Solutes that can cross the cellular membrane donot contribute to the final osmotic pressure gradient. Only thosedissolved species that do not cross the cell membrane will contribute toosmotic pressure differences and thus tonicity.

The term “hypertonic,” as generally used herein, refers to a solutionwith a higher concentration of solutes than is present on the inside ofthe cell. When a cell is immersed into a hypertonic solution, thetendency is for water to flow out of the cell in order to balance theconcentration of the solutes.

The term “hypotonic,” as generally used herein, refers to a solutionwith a lower concentration of solutes than is present on the inside ofthe cell. When a cell is immersed into a hypotonic solution, water flowsinto the cell in order to balance the concentration of the solutes.

The term “isotonic,” as generally used herein, refers to a solutionwherein the osmotic pressure gradient across the cell membrane isessentially balanced. An isotonic formulation is one which hasessentially the same osmotic pressure as human blood. Isotonicformulations will generally have an osmotic pressure from about 250mOsm/kg to 350 mOsm/kg.

The term “liquid formulation,” as used herein, is a protein that iseither supplied in an acceptable pharmaceutical diluent or one that isreconstituted in an acceptable pharmaceutical diluent prior toadministration to the patient.

The terms “branded” and “reference”, when used to refer to a protein orbiologic, are used interchangeably herein to mean the single biologicalproduct licensed under section 351(a) of the U.S. Public Health ServiceAct (42 U.S.C. § 262).

The term “biosimilar,” as used herein is generally used interchangeablywith “a generic equivalent” or “follow-on.” For example, a “biosimilarmAb” refers to a subsequent version of an innovator's mAb typically madeby a different company. “Biosimilar,” when used in reference to abranded protein or branded biologic, can refer to a biological productevaluated against the branded protein or branded biologic and licensedunder section 351(k) of the U.S. Public Health Service Act (42 U.S.C. §262). A biosimilar mAb can be one that satisfies one or more guidelinesadopted May 30, 2012 by the Committee for Medicinal Products for HumanUse (CHMP) of the European Medicines Agency and published by theEuropean Union as “Guideline on similar biological medicinal productscontaining monoclonal antibodies—non-clinical and clinical issues”(Document Reference EMA/CHMP/BMWP/403543/2010).

Biosimilars can be produced by microbial cells (prokaryotic,eukaryotic), cell lines of human or animal origin (e.g., mammalian,avian, insect), or tissues derived from animals or plants. Theexpression construct for a proposed biosimilar product will generallyencode the same primary amino acid sequence as its reference product.Minor modifications, such as N- or C-terminal truncations that will nothave an effect on safety, purity, or potency, may be present.

A biosimilar mAb is similar to the reference mAb physiochemically orbiologically, both in terms of safety and efficacy. The biosimilar mAbcan be evaluated against a reference mAb using one or more in vitrostudies including assays detailing binding to target antigen(s); bindingto isoforms of the Fc gamma receptors (FcγRI, FcγRII, and FcγRIII),FcRn, and complement (Clq); Fab-associated functions (e.g.neutralization of a soluble ligand, receptor activation or blockade); orFc-associated functions (e.g. antibody-dependent cell-mediatedcytotoxicity, complement-dependent cytotoxicity, complement activation).In vitro comparisons may be combined with in vivo data demonstratingsimilarity of pharmacokinetics, pharmacodynamics, and/or safety.Clinical evaluations of a biosimilar mAb against a reference mAb caninclude comparisons of pharmacokinetic properties (e.g. AUC_(0_inf),AUC_(0-t), C_(max), t_(max), C_(trough)); pharmacodynamic endpoints; orsimilarity of clinical efficacy (e.g. using randomized, parallel groupcomparative clinical trials). The quality comparison between abiosimilar mAb and a reference mAb can be evaluated using establishedprocedures, including those described in the “Guideline on similarbiological medicinal products containing biotechnology-derived proteinsas active substance: Quality issues” (EMEA/CHMP/BWP/49348/2005), and the“Guideline on development, production, characterization andspecifications for monoclonal antibodies and related substances”(EMEA/CHMP/BWP/157653/2007).

Differences between a biosimilar mAb and a reference mAb can includepost-translational modification, e.g. by attaching to the mAb otherbiochemical groups such as a phosphate, various lipids andcarbohydrates; by proteolytic cleavage following translation; bychanging the chemical nature of an amino acid (e.g., formylation); or bymany other mechanisms. Other post-translational modifications can be aconsequence of manufacturing process operations—for example, glycationmay occur with exposure of the product to reducing sugars. In othercases, storage conditions may be permissive for certain degradationpathways such as oxidation, deamidation, or aggregation. As all of theseproduct-related variants may be included in a biosimilar mAb.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic acids andbases, including inorganic acids and bases, and organic acids and bases.Suitable non-toxic acids include inorganic and organic acids such asacetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaricacid, and p-toluenesulfonic. Suitable positively charged counterionsinclude sodium, potassium, lithium, calcium and magnesium.

As used herein, the term “ionic liquid” refers to a crystalline oramorphous salt, zwitterion, or mixture thereof that is a liquid at ornear temperatures where most conventional salts are solids: at less than200° C., preferably less than 100° C. or more preferably less than 80°C. Some ionic liquids have melting temperatures around room temperature,e.g. between 10° C. and 40° C. or between 15° C. and 35° C. The term“zwitterion” is used herein to describe an overall neutrally chargedmolecule which carries formal positive and negative charges on differentchemical groups in the molecule. Examples of ionic liquids are describedin Riduan et al., Chem. Soc. Rev., 42:9055-9070, 2013; Rantwijk et al.,Chem. Rev., 107:2757-2785, 2007; Earle et al., Pure Appl. Chem.,72(7):1391-1398, 2000; and Sheldon et al., Green Chem., 4:147-151, 2002.

As used herein, a “water soluble organic dye” is an organic moleculehaving a molar solubility of at least 0.001 M at 25° C. and pH 7, andthat absorbs certain wavelengths of light, preferably in thevisible-to-infrared portion of the electromagnetic spectrum, whilepossibly transmitting or reflecting other wavelengths of light.

As used herein, the term “chalcogen” refers to Group 16 elements,including oxygen, sulfur and selenium, in any oxidation state. Forinstance, unless specified otherwise, the term “chalcogen” also includeSO₂.

The term “alkyl group,” as used herein, refers straight-chain,branched-chain and cyclic hydrocarbon groups. Unless specifiedotherwise, the term alkyl group embraces hydrocarbon groups containingone or more double or triple bonds. An alkyl group containing at leastone ring system is a “cycloalkyl” group. An alkyl group containing atleast one double bond is an “alkenyl group,” and an alkyl groupcontaining at least one triple bond is an “alkynyl group.”

“Aryl,” as used herein, refers to aromatic carbon ring systems,including fused ring systems. In an “aryl” group, each of the atoms thatform the ring is a carbon atom.

“Heteroaryl,” as used herein, refers to aromatic ring systems, includingfused ring systems, wherein at least one of the atoms that form the ringis a heteroatom.

“Heterocycle” as used hererin, refers to ring systems that, includingfused ring systems, are not aromatic, wherein at least one of the atomsthat forms the ring is a heteroatom.

As used herein, a “heteroatom” is any non-carbon or non-hydrogen atom.Preferred heteroatoms include oxygen, sulfur, and nitrogen. Exemplaryheteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, and xanthenyl.

The term “organophosphate” herein refers to a compound containing one ormore phosphoryl groups at least one of which is covalently connected toan organic group through a phosphoester bond. Organophosphates can benaturally occurring or non-naturally occurring, synthetic, orsemi-synthetic.

The term “nucleobase”, as used herein, refers broadly to substituted andunsubstituted nitrogen-containing heteroaromatic rings, preferablyhaving both a hydrogen-bond donating group and a hydrogen-bond receivinggroup and capable of forming Watson-Crick hydrogen bonds with acomplimentary nucleobase. Nucleobases include naturally occurringnucleobases and non-naturally occurring nucleobases. Non-naturallyoccurring nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-methyl pyrimidines such as 5-methylcytosine,5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as wellas synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N⁶-(6-aminohexyl)adenine, and 2,6-diaminopurine.Nucleobases include purine and purine bases such as adenine, guanine,hypoxanthine, xanthine, and 7-methylguanine. Nucleobases includepyrimidine and pyrimidine bases such as thymine, cytosine, uracil,5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

Certain organophosphates contain acidic or basic functional groups.Whether or not these functional groups are fully or partially ionizeddepends on the pH of the formulation they are in. Unless otherwisespecified, reference to a formulation containing an organophosphatehaving an ionizable functional group includes both the parent compoundand any possible ionized states.

The term “nucleoside” is used herein to refer to any compound in which anucleobase is covalently coupled to a five-carbon sugar, preferably inits cyclic form. Five-carbon sugars include ribose, deoxyribose,arabinose, xylose, lyxose, and derivatives thereof.

The term “nucleotide” is used herein to refer to any compounds in whicha nucleoside is covalently coupled at one or more positions on the sugarto a phosphate group or polyphosphate group, e.g. a diphosphate ortriphosphate group.

II. Formulations

Biocompatible, low-viscosity protein solutions, such as those of mAbs,can be used to deliver therapeutically effective amounts of proteins involumes useful for subcutaneous (SC) and intramuscular (IM) injections,typically less than or about 2 mL for SC and less than or about 5 mL forIM, more preferably less than or about 1 mL for SC and less than orabout 3 mL for IM. The proteins can generally have any molecular weight,although in some embodiments high-molecular-weight proteins arepreferred. In other embodiments the proteins are low-molecular-weightproteins.

Formulations may have protein concentrations between about 10 mg/mL andabout 5,000 mg/mL. The formulations, including mAb formulations, mayhave a protein concentration greater than 100 mg/mL, preferably greaterthan 150 mg/mL, more preferably greater than about 175 mg/ml, preferablygreater even more than about 200 mg/mL, even more preferably greaterthan about 225 mg/mL, even more preferably greater than about 250 mg/mL,and most preferably greater than or about 300 mg/mL. In the absence ofan organophosphate, the viscosity of a protein formulation increasesexponentially as the concentration is increased. Such proteinformulations, in the absence of an organophosphate, may have viscositiesgreater than 100 cP, greater than 150 cP, greater than 200 cP, greaterthan 300 cP, greater than 500 cP, or even greater than 1,000 cP, whenmeasured at 25° C. Such formulations are often unsuitable for SC or IMinjection. The use of one or more viscosity-reducing organophosphatespermits the preparation of formulations having a viscosity less than orabout 100 cP, preferably less than or about 75 cP, more preferably lessthan or about 50 cP, less than or about less than or about 30 cP, lessthan or about 20 cP, or even most preferably less than or about 10 cP,when measured at 25° C.

Although the viscosity-reducing organophosphates may be used to lowerthe viscosity of concentrated protein formulations, they may be used inless-concentrated formulations as well. In some embodiments,formulations may have protein concentrations between about 10 mg/mL andabout 100 mg/mL. The formulations may have a protein concentrationgreater than about 20 mg/mL, greater than about 40 mg/mL, or greaterthan about 80 mg/mL.

For certain proteins, formulations not having a viscosity-reducingorganophosphate may have viscosities greater than about 20 cP, greaterthan about 50 cP, or greater than about 80 cP. The use of one or moreviscosity-reducing organophosphates permits the preparation offormulations having a viscosity less than or about 80 cP, preferablyless than or about 50 cP, even more preferably less than about 20 cP, ormost preferably less than or about 10 cP, when measured at 25° C.

In some embodiments, the aqueous protein formulations have a viscositythat is at least about 30% less than the analogous formulation withoutthe viscosity-reducing organophosphate(s), when measured under the sameconditions. In other embodiments, the formulations have a viscosity thatis 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or evenmore than 90% less than the analogous formulation without theviscosity-reducing organophosphate(s). In a preferred embodiment, theformulation contains a therapeutically effective amount of the one ormore high-molecular-weight proteins, such as mAbs, in a volume of lessthan about 2 mL, preferably less than about 1 mL, or more preferablyless than about 0.75 mL.

The reduced-viscosity formulations have improved injectability andrequire less injection force compared to the analogous formulationwithout the viscosity-reducing organophosphate (e.g., in a sodiumphosphate buffer) under otherwise the same conditions. In someembodiments, the force of injection is decreased by more than about 20%,more than about 30%, more than about 40%, more than about 50%, or morethan about 2 fold, as compared to standard formulations without theviscosity-reducing organophosphate(s) under otherwise the same injectionconditions. In some embodiments, the formulations possess “Newtonianflow characteristics,” defined as having viscosity which issubstantially independent of shear rate. The protein formulations can bereadily injected through needles of about 18-32 gauge. Preferred needlegauges for the delivery of the low-viscosity formulations include 27,29, and 31 gauge, optionally thin walled.

The formulations may contain one or more additional excipients, such asbuffers, surfactants, sugars and sugar alcohols, other polyols,preservatives, antioxidants, and chelating agents. The formulations havea pH and osmolarity suitable for administration without causingsignificant adverse side effects. In some embodiments, the concentrated,low-viscosity formulations have a pH between 5 and 8, between 5.5 and7.6, between 6.0 and 7.6, or between 5.5 and 6.5.

The low-viscosity protein formulations can allow for greater flexibilityin formulation development. The low-viscosity formulations can exhibitchanges in viscosity that are less dependent upon the proteinconcentration as compared to the otherwise same formulation without theviscosity-reducing organophosphate. The low-viscosity proteinformulations can allow for increased concentrations and decreased dosagefrequencies of the protein. In some embodiments the low-viscosityprotein formulations contain 2 or more, 3 or more, or 4 or moredifferent proteins. For example, combinations of 2 or more mAbs can beprovided in a single low-viscosity protein formulation.

Because protein (such as mAb) formulations may be administered topatients at higher protein concentrations than otherwise similar proteinformulations not containing a viscosity-reducing organophosphate, thedosing frequency of the protein can be reduced. For instance, proteinspreviously requiring once daily administration may be administered onceevery two days, every three days, or even less frequently when theproteins are formulated with viscosity-reducing organophosphates.Proteins which currently require multiple administrations on the sameday (either at the same time or at different times of the day) may beadministered in fewer injections per day. In some instances, thefrequency may be reduced to a single injection once a day. By increasingthe dosage administered per injection multiple-fold the dosing frequencycan be decreased, for example from once every 2 weeks to once every 6weeks.

In some embodiments, the liquid formulations herein have a physiologicalosmolarity, for example, between about 280 mOsm/L to about 310 mOsm/L.In some embodiments, the liquid formulations have an osmolarity greaterthan about 250 mOsm/L, greater than about 300 mOsm/L, greater than about350 mOsm/L, greater than about 400 mOsm/L, or greater than about 500mOsm/L. In some embodiments, the formulations have an osmolarity ofabout 200 mOsm/L to about 2,000 mOsm/L or about 300 mOsm/L to about1,000 mOsm/L. In some embodiments, the liquid formulations areessentially isotonic to human blood. The liquid formulations can in somecases be hypertonic.

The additives, including the viscosity-reducing organophosphates, can beincluded in any amount to achieve the desired viscosity levels of theliquid formulation, as long as the amounts are not toxic or otherwiseharmful, and do not substantially interfere with the chemical and/orphysical stability of the formulation. The viscosity-reducingorganophosphate(s) in some embodiments can be independently present in aconcentration less than about 1.0 M, preferably less than about 0.50 M,less than or equal to about 0.30 M or less than or equal to 0.15 M.Especially preferred concentrations include about 0.10 M and about 0.30M. For some embodiments having two or more viscosity-reducingorganophosphates, the compounds are preferably, but not necessarily,present at the same concentration.

The viscosity-reducing organophosphates permit faster reconstitution ofa lyophilized dosage unit. The dosage unit is a lyophilized cake ofprotein, viscosity-reducing organophosphate and other excipients, towhich water, saline or another pharmaceutically acceptable fluid isadded. In the absence of viscosity-reducing organophosphates, periods of10 minutes or more are often required in order to completely dissolvethe lyophilized cake at high protein concentration. When the lyophilizedcake contains one or more viscosity-reducing organophosphates, theperiod required to completely dissolve the cake is often reduced by afactor of two, five or even ten. In certain embodiments, less than oneminute is required to completely dissolve a lyophilized cake containinggreater than or about 150, 200 or even 300 mg/mL of protein.

The low-viscosity protein formulations allow for greater flexibility informulation development. The low-viscosity formulations exhibit aviscosity that changes less with increasing protein concentrations ascompared to the otherwise same formulation without theviscosity-reducing organophosphate(s). The low-viscosity proteinformulations exhibit a decreased viscosity gradient as compared to theotherwise same formulation without the viscosity-reducingorganophosphate.

The viscosity gradient of the protein formulation may be 2-fold less,3-fold less, or even more than 3-fold less than the viscosity gradientof the otherwise same protein formulation without the viscosity-reducingorganophosphate(s). The viscosity gradient of the protein formulationmay be less than 2.0 cP mL/mg, less than 1.5 cP mL/mg, less than 1.0 cPmL/mg, less than 0.8 cP mL/mg, less than 0.6 cP mL/mg, or less than 0.2cP mL/mg for a protein formulation having a protein concentrationbetween 10 mg/mL and 2,000 mg/mL. By reducing the viscosity gradient ofthe formulation, the protein concentration can be increased to a greaterdegree before an exponential increase in viscosity is observed

A. Proteins

Any protein can be formulated, including recombinant, isolated, orsynthetic proteins, glycoproteins, or lipoproteins. These may beantibodies (including antibody fragments and recombinant antibodies),enzymes, growth factors or hormones, immunomodifiers, antiinfectives,antiproliferatives, vaccines, or other therapeutic, prophylactic, ordiagnostic proteins. In certain embodiments, the protein has a molecularweight greater than about 150 kDa, greater than 160 kDa, greater than170 kDa, greater than 180 kDa, greater than 190 kDa or even greater than200 kDa.

In certain embodiments, the protein can be a PEGylated protein. The term“PEGylated protein,” as used herein, refers to a protein having one ormore poly(ethylene glycol) or other stealth polymer groups covalentlyattached thereto, optionally through a chemical linker that may bedifferent from the one or more polymer groups. PEGylated proteins arecharacterized by their typically reduced renal filtration, decreaseduptake by the reticuloendothelial system, and diminished enzymaticdegradation leading to, for example, prolonged half-lives and enhancedbioavailability. Stealth polymers include poly(ethylene glycol);poly(propylene glycol); poly(amino acid) polymers such as poly(glutamicacid), poly(hydroxyethyl-L-asparagine), andpoly(hydroxethyl-L-glutamine); poly(glycerol); poly(2-oxazoline)polymers such as poly(2-methyl-2-oxazoline) andpoly(2-ethyl-2-oxazoline); poly(acrylamide); poly(vinylpyrrolidone);poly(N-(2-hydroxypropyl)methacrylamide); and copolymers and mixturesthereof. In preferred embodiments the stealth polymer in a PEGylatedprotein is poly(ethylene glycol) or a copolymer thereof. PEGylatedproteins can be randomly PEGylated, i.e. having one or more stealthpolymers covalently attached at non-specific site(s) on the protein, orcan be PEGylated in a site-specific manner by covalently attaching thestealth polymer to specific site(s) on the protein. Site-specificPEGylation can be accomplished, for example, using activated stealthpolymers having one or more reactive functional groups. Examples aredescribed, for instance, in Hoffman et al., Progress in Polymer Science,32:922-932, 2007.

In the preferred embodiment, the protein is high-molecular-weight and anantibody, most preferably a mAb, and has a high viscosity in aqueousbuffered solution when concentrated sufficiently to inject atherapeutically effective amount in a volume not exceeding 1.0 to 2.0 mLfor SC and 3.0 to 5.0 mL for IM administration. High-molecular-weightproteins can include those described in Scolnik, mAbs 1:179-184, 2009;Beck, mAbs 3:107-110, 2011; Baumann, Curr. Drug Meth. 7:15-21, 2006; orFederici, Biologicals 41:131-147, 2013. The proteins for use in theformulations described herein are preferably essentially pure andessentially homogeneous (i.e., substantially free from contaminatingproteins and/or irreversible aggregates thereof).

Preferred mAbs herein include natalizumab (TYSABRI®), cetuximab(ERBITUX®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), infliximab(REMICADE®), rituximab (RITUXAN®), panitumumab (VECTIBIX®), ofatumumab(ARZERRA®), and biosimilars thereof. Exemplary high-molecular-weightproteins can include tocilizumab (ACTEMRA®), alemtuzumab (marketed underseveral trade names), brodalumab (developed by Amgen, Inc (“Amgen”)),denosumab (PROLIA® and XGEVA®), and biosimilars thereof.

Exemplary molecular targets for antibodies described herein include CDproteins, such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HERreceptor family such as the EGF receptor, HER2, HER3 or HER4 receptor;cell adhesion molecules, such as LFA-1, Mol, p150,95, VLA-4, ICAM-1,VCAM, and av/r33 integrin, including either α or β subunits thereof(e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors,such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity(OB) receptor; protein C; PCSK9; etc.

Antibody Therapeutics Currently on the Market

Many protein therapeutics currently on the market, especially antibodiesas defined herein, are administered via IV infusions due to high dosingrequirements. Formulations can include one of the antibody therapeuticscurrently on the market or a biosimilar thereof. Some proteintherapeutics currently on the market are not high-molecular-weight, butare still administered via IV infusion because high doses are needed fortherapeutic efficacy. In some embodiments, liquid formulations areprovided of these low-molecular-weight proteins as defined herein withconcentrations to deliver therapeutically effective amounts for SC or IMinjections.

Antibody therapeutics currently on the market include belimumab(BENLYSTA®), golimumab (SIMPONI ARIA®), abciximab (REOPRO®), thecombination of tositumomab and iodine-131 tositumomab, marketed asBEXXAR®, alemtuzumab (CAMPATH®), palivizumab (SYNAGIS®), basiliximab(SIMULECT®), ado-trastuzumab emtansine (KADCYLA®), pertuzumab(PERJETA®), capromab pendetide (PROSTASCINT KIT®), caclizumab(ZENAPAX®), ibritumomab tiuxetan (ZEVALIN®), eculizumab (SOLIRIS®),ipilimumab (YERVOY®), muromonab-CD3 (ORTHOCLONE OKT3®), raxibacumab,nimotuzumab (THERACIM®), brentuximab vedotin (ADCETRIS®), adalimumab(HUMIRA®), golimumab (SIMPONI®), palivizumab (SYNAGIS®), omalizumab(XOLAIR®), and ustekinumab (STELARA®).

Natalizumab, a humanized mAb against the cell adhesion moleculea4-integrin, is used in the treatment of multiple sclerosis and Crohn'sdisease. Previously marketed under the trade name ANTEGREN®, natalizumabis currently co-marketed as TYSABRI® by Biogen Idec (“Biogen”) and ElanCorp. (“Elan”) TYSABRT® is produced in murine myeloma cells. Each 15 mLdose contains 300 mg natalizumab; 123 mg sodium chloride, USP; 17.0 mgsodium phosphate, monobasic, monohydrate, USP; 7.24 mg sodium phosphate,dibasic, heptahydrate, USP; 3.0 mg polysorbate 80, USP/NF, in water forIV injection, USP at pH 6.1. Natalizumab is typically administered bymonthly intravenous (IV) infusions and has been proven effective intreating the symptoms of both multiple sclerosis and Crohn's disease, aswell as for preventing relapse, vision loss, cognitive decline, andsignificantly improving patient's quality of life.

As used herein, the term “natalizumab” includes the mAb against the celladhesion molecule α4-integrin known under the InternationalNonproprietary Name “NATALIZUMAB” or an antigen binding portion thereof.Natalizumab includes antibodies described in U.S. Pat. Nos. 5,840,299,6,033,665, 6,602,503, 5,168,062, 5,385,839, and 5,730,978. Natalizumabincludes the active agent in products marketed under the trade nameTYSABRI® by Biogen Idec and Elan Corporation or a biosimilar productthereof.

Cetuximab is an epidermal growth factor receptor (EGFR) inhibitor usedfor the treatment of metastatic colorectal cancer and head and neckcancer. Cetuximab is a chimeric (mouse/human) mAb typically given by IVinfusion. Cetuximab is marketed for IV use only under the trade nameERBITUX® by Bristol-Myers Squibb Company (North America; “Bristol-MyersSquibb”), Eli Lilly and Company (North America; “Eli Lilly”), and MerckKGaA. ERBITUX® is produced in mammalian (murine myeloma) cell culture.Each single-use, 50-mL vial of ERBITUX® contains 100 mg of cetuximab ata concentration of 2 mg/mL and is formulated in a preservative-freesolution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodiumphosphate dibasic heptahydrate, 0.42 mg/mL sodium phosphate monobasicmonohydrate, and water for IV Injection, USP.

Cetuximab is indicated for the treatment of patients with epidermalgrowth factor receptor (EGFR)-expressing, KRAS wild-type metastaticcolorectal cancer (mCRC), in combination with chemotherapy, and as asingle agent in patients who have failed oxaliplatin- andirinotecan-based therapy or who are intolerant to irinotecan. Cetuximabis indicated for the treatment of patients with squamous cell carcinomaof the head and neck in combination with platinum-based chemotherapy forthe first-line treatment of recurrent and/or metastatic disease and incombination with radiation therapy for locally advanced disease.Approximately 75% of patients with metastatic colorectal cancer have anEGFR-expressing tumor and are, therefore, considered eligible fortreatment with cetuximab or panitumumab, according to FDA guidelines.

As used herein, the term “cetuximab” includes the mAb known under theInternational Nonproprietary Name “CETUXIMAB” or an antigen bindingportion thereof. Cetuximab includes antibodies described in U.S. Pat.No. 6,217,866. Cetuximab includes the active agent in products marketedunder the trade name ERBITUX® and biosimilar products thereof.Biosimilars of ERBITUX® can include those currently being developed byAmgen, AlphaMab Co., Ltd. (“AlphaMab”), and Actavis plc (“Actavis”).

Bevacizumab, a humanized mAb that inhibits vascular endothelial growthfactor A (VEGF-A), acts as an anti-angiogenic agent. It is marketedunder the trade name AVASTIN® by Genentech, Inc. (“Genentech”) and F.Hoffmann-La Roche, LTD (“Roche”). It is licensed to treat variouscancers, including colorectal, lung, breast (outside the U.S.A.),glioblastoma (U.S.A. only), kidney and ovarian. AVASTIN® was approved bythe FDA in 2004 for use in metastatic colorectal cancer when used withstandard chemotherapy treatment (as first-line treatment) and with5-fluorouracil-based therapy for second-line metastatic colorectalcancer. In 2006, the FDA approved AVASTIN® for use in first-lineadvanced non-squamous non-small cell lung cancer in combination withcarboplatin/paclitaxel chemotherapy. AVASTIN® is given as an IV infusionevery three weeks at the dose of either 15 mg/kg or 7.5 mg/kg. Thehigher dose is usually given with carboplatin-based chemotherapy,whereas the lower dose is given with cisplatin-based chemotherapy. In2009, the FDA approved AVASTIN® for use in metastatic renal cellcarcinoma (a form of kidney cancer). The FDA also granted acceleratedapproval of AVASTIN® for the treatment of recurrent glioblastomamultiforme in 2009. Treatment for initial growth is still in phase IIIclinical trial.

The National Comprehensive Cancer Network (“NCCN”) recommendsbevacizumab as standard first-line treatment in combination with anyplatinum-based chemotherapy, followed by maintenance bevacizumab untildisease progression. The NCCN updated its Clinical Practice Guidelinesfor Oncology (NCCN Guidelines) for Breast Cancer in 2010 to affirm therecommendation regarding the use of bevacizumab (AVASTIN®,Genentech/Roche) in the treatment of metastatic breast cancer.

As used herein, the term “bevacizumab” includes the mAb that inhibitsvascular endothelial growth factor A (VEGF-A) known under theInternational Nonproprietary Name/Common Name “BEVACIZUMAB” or anantigen binding portion thereof. Bevacizumab is described in U.S. Pat.No. 6,054,297. Bevacizumab includes the active agent in productsmarketed under the trade name AVASTIN® and biosimilar products thereof.Biosimilars of AVASTIN® can include those currently being developed byAmgen, Actavis, AlphaMab, and Pfizer, Inc (“Pfizer”). Biosimilars ofAVASTIN® can include the biosimilar known as BCD-021 produced by Biocadand currently in clinical trials in the U.S.

Trastuzumab is a mAb that interferes with the HER2/neu receptor.Trastuzumab is marketed under the trade name HERCEPTIN® by Genentech,Inc. HERCEPTIN® is produced by a mammalian cell (Chinese Hamster Ovary(CHO)) line. HERCEPTIN® is a sterile, white to pale-yellow,preservative-free lyophilized powder for IV administration. EachHERCEPTIN® vial contains 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4mg L-histidine, 400 mg a,a-trehalose dihydrate, and 1.8 mg polysorbate20, USP. Reconstitution with 20 mL water yields a multi-dose solutioncontaining 21 mg/mL trastuzumab. HERCEPTIN® is currently administeredvia IV infusion as often as weekly and at a dosage ranging from about 2mg/kg to about 8 mg/kg.

Trastuzumab is mainly used to treat certain breast cancers. The HER2gene is amplified in 20-30% of early-stage breast cancers, which makesit overexpress epidermal growth factor (EGF) receptors in the cellmembrane. Trastuzumab is generally administered as a maintenance therapyfor patients with HER2-positive breast cancer, typically for one yearpost-chemotherapy. Trastuzumab is currently administered via IV infusionas often as weekly and at a dosage ranging from about 2 mg/kg to about 8mg/kg.

As used herein, the term “trastuzumab” includes the mAb that interfereswith the HER2/neu receptor known under the International NonproprietaryName/Common Name “TRASTUZUMAB” or an antigen binding portion thereof.Trastuzumab is described in U.S. Pat. No. 5,821,337. Trastuzumabincludes the active agent in products marketed under the trade nameHERCEPTIN® and biosimilars thereof. The term “trastuzumab” includes theactive agent in biosimilar HERCEPTIN® products marketed under the tradenames HERTRAZ® by Mylan, Inc. (“Mylan”) and CANMAB® by Biocon, Ltd.(“Biocon”). Trastuzumab can include the active agent in biosimilarHERCEPTIN® products being developed by Amgen and by PlantFormCorporation, Canada.

Infliximab is a mAb against tumor necrosis factor alpha (TNF-α) used totreat autoimmune diseases. It is marketed under the trade name REMICADE®by Janssen Global Services, LLC (“Janssen”) in the U.S., MitsubishiTanabe Pharma in Japan, Xian Janssen in China, and Merck & Co (“Merck”);elsewhere. Infliximab is a chimeric mouse/human monoclonal antibody witha high molecular weight of approximately 144 kDa. In some embodiments,the formulations contain a biosimilar of REMICADE®, such as REMSIMA™ orINFLECTRA™. Both REMSIMA™, developed by Celltrion, Inc. (“Celltrion”),and INFLECTRA™, developed by Hospira Inc, UK, have been recommended forregulatory approval in Europe. Celltrion has submitted a filing forREMSIMA™ to the FDA. Infliximab is currently administered via IVinfusion at doses ranging from about 3 mg/kg to about 10 mg/kg.

Infliximab contains approximately 30% murine variable region amino acidsequence, which confers antigen-binding specificity to human TNFα. Theremaining 70% correspond to a human IgG1 heavy chain constant region anda human kappa light chain constant region. Infliximab has high affinityfor human TNFα, which is a cytokine with multiple biologic actionsincluding mediation of inflammatory responses and modulation of theimmune system.

Infliximab is a recombinant antibody generally produced and secretedfrom mouse myeloma cells (SP2/0 cells). The antibody is currentlymanufactured by continuous perfusion cell culture. The infliximabmonoclonal antibody is expressed using chimeric antibody genesconsisting of the variable region sequences cloned from the murineanti-TNFα hybridoma A2, and human antibody constant region sequencessupplied by the plasmid expression vectors. Generation of the murineanti-TNF a hybridoma is performed by immunization of BALB/c mice withpurified recombinant human TNFα. The heavy and light chain vectorconstructs are linearized and transfected into the Sp2/0 cells byelectroporation. Standard purification steps can include chromatographicpurification, viral inactivation, nanofiltration, andultrafiltration/diafiltration.

As used herein, the term “infliximab” includes the chimeric mouse/humanmonoclonal antibody known under the International Nonproprietary Name“INFLIXIMAB” or an antigen binding portion thereof. Infliximabneutralizes the biological activity of TNFα by binding with highaffinity to the soluble and transmembrane forms of TNFα and inhibitsbinding of TNFα with its receptors. Infliximab is described in U.S. Pat.No. 5,698,195. The term “Infliximab” includes the active agent inproducts marketed or proposed to be marketed under the trade namesREMICADE® by multiple entities; REMSIMA™ by Celltrion and INFLECTRA™ byHospira, Inc (“Hospira”). Infliximab is supplied as a sterilelyophilized cake for reconstitution and dilution. Each vial ofinfliximab contains 100 mg infliximab and excipients such as monobasicsodium phosphate monohydrate, dibasic sodium phosphate dihydrate,sucrose, and polysorbate 80.

Denosumab (PROLIA® and XGEVA®) is a human mAb—and the first RANKLinhibitor—approved for use in postmenopausal women with risk ofosteoporosis and patients with bone metastases from solid tumors.Denosumab is in Phase II trials for the treatment of rheumatoidarthritis.

Panitumumab is a fully human mAb approved by the FDA for treatment ofEGFR-expressing metastatic cancer with disease progression. Panitumumabis marketed under the trade name VECTIBIX® by Amgen. VECTIBIX® ispackaged as a 20 mg/ml panitumumab concentrate in 5 ml, 10 ml, and 15 mlvials for IV infusion. When prepared according to the packaginginstructions, the final panitumumab concentration does not exceed 10mg/ml. VECTIBIX® is administered at a dosage of 6 mg/kg every 14 days asan intravenous infusion. As used herein, the term “panitumumab” includesthe anti-human epidermal growth factor receptor known by theInternational Nonproprietary Name “PANITUMUMAB.” The term “panitumumab”includes the active agent in products marketed under the trade nameVECTIBIX® by Amgen and biosimilars thereof. The term “panitumumab”includes monoclonal antibodies described in U.S. Pat. No. 6,235,883. Theterm “panitumumab” includes the active agent in biosimilar VECTIBIX®products, including biosimilar VECTIBIX® being developed by BioXpress,SA (“BioXpress”).

Belimumab (BENLYSTA®) is a human mAb with a molecular weight of about151.8 kDa that inhibits B-cell activating factor (BAFF). Belimumab isapproved in the United States, Canada, and Europe for treatment ofsystemic lupus erythematosus. Belimumab is currently administered tolupus patients by IV infusion at a 10 mg/kg dosage. Ahigh-molecular-weight, low-viscosity protein formulation can includeBelimumab, preferably in a concentration of about 400 mg/mL to about1,000 mg/mL. The preferred ranges are calculated based upon body weightof 40-100 kg (approximately 80-220 lbs) in a 1 mL volume.

Abciximab (REOPRO®) is manufactured by Janssen Biologics BV anddistributed by Eli Lilly & Company (“Eli Lilly”). Abciximab is a Fabfragment of the chimeric human-murine monoclonal antibody 7E3. Abciximabbinds to the glycoprotein (GP) IIb/IIIa receptor of human platelets andinhibits platelet aggregation by preventing the binding of fibrinogen,von Willebrand factor, and other adhesive molecules. It also binds tovitronectin (αvβ3) receptor found on platelets and vessel wallendothelial and smooth muscle cells. Abciximab is a platelet aggregationinhibitor mainly used during and after coronary artery procedures.Abciximab is administered via IV infusion, first in a bolus of 0.25mg/kg and followed by continuous IV infusion of 0.125 mcg/kg/minute for12 hours.

Tositumomab (BEXXAR®) is a drug for the treatment of follicularlymphoma. It is an IgG2a anti-CD20 mAb derived from immortalized mousecells. Tositumomab is administered in sequential infusions: cold mAbfollowed by iodine (131I) tositumomab, the same antibody covalentlybound to the radionuclide iodine-131. Clinical trials have establishedthe efficacy of the tositumomab/iodine tositumomab regimen in patientswith relapsed refractory follicular lymphoma. BEXXAR® is currentlyadministered at a dose of 450 mg via IV infusion.

Alemtuzumab (marketed as CAMPATH®, MABCAMPATH®, or CAMPATH-1H® andcurrently under further development as LEMTRADA®) is a mAb used in thetreatment of chronic lymphocytic leukemia (CLL), cutaneous T-celllymphoma (CTCL), and T-cell lymphoma. It is also used under clinicaltrial protocols for treatment of some autoimmune diseases, such asmultiple sclerosis. Alemtuzumab has a weight of approximately 145.5 kDa.It is administered in daily IV infusions of 30 mg for patients withB-cell chronic lymphocytic leukemia.

Palivizumab (SYNAGIS®) is a humanized mAb directed against an epitope inthe A antigenic site of the F protein of respiratory syncytial virus. Intwo Phase III clinical trials in the pediatric population, palivizumabreduced the risk of hospitalization due to respiratory syncytial virusinfection by 55% and 45%. Palivizumab is dosed once a month via IMinjection of 15 mg/kg.

Ofatumumab is a human anti-CD20 mAb which appears to inhibit early-stageB lymphocyte activation. Ofatumumab is marketed under the trade nameARZERRA® by GlaxoSmithKline, plc (“GlaxoSmithKline”). ARZERRA® isdistributed in single-use vials containing 100 mg/5 mL and 1,000 mg/50mL ofatumumab for IV infusion. Ofatumumab is FDA-approved for treatingchronic lymphocytic leukemia and has also shown potential in treatingFollicular non-Hodgkin's lymphoma, Diffuse large B cell lymphoma,rheumatoid arthritis, and relapsing remitting multiple sclerosis.Ofatumumab has a molecular weight of about 149 kDa. It is currentlyadministered by IV infusion at an initial dose of 300 mg, followed byweekly infusions of 2,000 mg. As used herein, the term “ofatumumab”includes the anti-CD20 mAb known by the International NonproprietaryName “OFATUMUMAB.” The term “ofatumumab” includes the active agent inproducts marketed under the trade name ARZERRA® and biosimilars thereof.The term “ofatumumab” includes the active agent in biosimilar ARZERRA®products being developed by BioExpress. High-molecular-weight,low-viscosity liquid protein formulations can include ofatumumab,preferably in a concentration of about 300 mg/mL to about 2,000 mg/mL.

Trastuzumab emtansine (in the U.S., ado-trastuzumab emtansine, marketedas KADCYLA®) is an antibody-drug conjugate consisting of the mAbtrastuzumab linked to the cytotoxic agent mertansine (DM1®).Trastuzumab, described above, stops growth of cancer cells by binding tothe HER2/neu receptor, whereas mertansine enters cells and destroys themby binding to tubulin. In the United States, trastuzumab emtansine wasapproved specifically for treatment of recurring HER2-positivemetastatic breast cancer. Multiple Phase III trials of trastuzumabemtansine are planned or ongoing in 2014. Trastuzumab emtansine iscurrently administered by IV infusion of 3.6 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includetrastuzumab emtansine, preferably in a concentration of about 144 mg/mLto about 360 mg/mL.

Pertuzumab (PERJETA®) is a mAb that inhibits HER2 dimerization.Pertuzumab received FDA approval for the treatment of HER2-positivemetastatic breast cancer in 2012. The currently recommended dosage ofPertuzumab is 420 mg to 840 mg by IV infusion. High-molecular-weight,low-viscosity liquid formulations can include pertuzumab, preferably ina concentration of about 420 mg/mL to about 840 mg/mL.

Daclizumab is a humanized anti-CD25 mAb and is used to prevent rejectionin organ transplantation, especially in kidney transplants. The drug isalso under investigation for the treatment of multiple sclerosis.Daclizumab has a molecular weight of about 143 kDa. Daclizumab wasmarketed in the U.S. by Hoffmann-La Roche, Ltd. (“Roche”) as ZENAPAX®and administered by IV infusion of 1 mg/kg. Daclizumab High-YieldProcess (DAC HYP; BIIB019; Biogen Idec (“Biogen”) and AbbVie, Inc.(“AbbVie”)) is in phase III clinical trials as a 150 mg, once-monthlysubcutaneous injection to treat relapsing, remitting multiple-sclerosis.High-molecular-weight, low-viscosity liquid formulations can includedaclizumab, preferably in a concentration of about 40 mg/mL to about 300mg/mL.

Eculizumab (SOLIRIS®) is a humanized mAb approved for the treatment ofrare blood diseases, such as paroxysmal nocturnal hemoglobinuria andatypical hemolytic uremic syndrome. Eculizumab, with a molecular weightof about 148 kDa, is being developed by Alexion Pharmaceuticals, Inc(“Alexion”). It is administered by IV infusion in the amount of about600 mg to about 1,200 mg. High-molecular-weight, low-viscosity liquidformulations can include eculizumab, preferably in a concentration ofabout 500 mg/mL to about 1,200 mg/mL.

Tocilizumab (ACTEMRA®) is a humanized mAb against the interleukin-6receptor. It is an immunosuppressive drug, mainly for the treatment ofrheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, asevere form of RA in children. Tocilizumab is commonly administered byIV infusion in doses of about 6 mg/kg to about 8 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includetocilizumab, preferably in a concentration of about 240 mg/mL to about800 mg/mL.

Rituximab (RITUXAN®) is a chimeric anti-CD20 mAb used to treat a varietyof diseases characterized by excessive numbers of B cells, overactive Bcells, or dysfunctional B cells. Rituximab is used to treat cancers ofthe white blood system, such as leukemias and lymphomas, includingHodgkin's lymphoma and its lymphocyte-predominant subtype. It has beenshown to be an effective rheumatoid arthritis treatment. Rituximab iswidely used off-label to treat difficult cases of multiple sclerosis,systemic lupus erythematosus, and autoimmune anemias.

Rituximab is jointly marketed in the U.S. under the trade name RITUXAN®by Biogen and Genentech and outside the U.S. under the trade nameMABTHERA® by Roche. RITUXAN® is distributed in single-use vialscontaining 100 mg/10 mL and 500 mg/50 mL. RITUXAN® is typicallyadministered by IV infusion of about 375 mg/m². The term “rituximab,” asused herein, includes the anti-CD20 mAb known under the InternationalNonproprietary Name/Common Name “RITUXIMAB.” Rituximab includes mAbsdescribed in U.S. Pat. No. 5,736,137. Rituximab includes the activeagent in products marketed under the trade name RITUXAN® and MABTHERA®and biosimilars thereof.

High-molecular-weight, low-viscosity liquid formulations can includerituximab, preferably in a concentration of about 475 mg/mL to about 875mg/mL (approximated using a body surface area range of 1.3 to 2.3 squaremeters, derived from the Mosteller formula for persons ranging from 5ft, 40 kg to 6 ft, 100 kg). Concentrations are calculated for a 1 mLformulation.

Ipilimumab is a human mAb developed by Bristol-Myers Squibb Company(“Bristol-Myers Squibb”). Marketed as YERVOY®, it is used for thetreatment of melanoma and is also undergoing clinical trials for thetreatment of non-small cell lung carcinoma (NSCLC), small cell lungcancer (SCLC), and metastatic hormone-refractory prostate cancer.Ipilimumab is currently administered by IV infusion of 3 mg/kg.High-molecular-weight, low-viscosity liquid formulations can includeipilimumab, preferably in a concentration of about 120 mg/mL to about300 mg/mL.

Raxibacumab (ABthrax®) is a human mAb intended for the prophylaxis andtreatment of inhaled anthrax. It is currently administered by IVinfusion. The suggested dosage in adults and children over 50 kg is 40mg/kg. High-molecular-weight, low-viscosity liquid formulations caninclude raxibacumab, preferably in a concentration of about 1,000 mg/mLto about 4,000 mg/mL.

Nimotuzumab (THERACIM®, BIOMAB EGFR®, THERALOC®, CIMAher®) is ahumanized mAb with a molecular weight of about 151 kDa used to treatsquamous cell carcinomas of the head and neck, recurrent or refractoryhigh-grade malignant glioma, anaplastic astrocytomas, glioblastomas, anddiffuse intrinsic pontine glioma. Nimotuzumab is typically administeredby IV infusion of about 200 mg weekly. High-molecular-weight,low-viscosity liquid formulations can include nimotuzumab, preferably ina concentration of about 200 mg/mL.

Brentuximab vedotin (ADCETRTS®) is an antibody-drug conjugate directedto the protein CD30, expressed in classical Hodgkin's lymphoma andsystemic anaplastic large cell lymphoma. It is administered by IVinfusion of about 1.8 mg/kg. High-molecular-weight, low-viscosity liquidformulations can include brentuximab vedotin, preferably in aconcentration of about 80 mg/mL to about 200 mg/mL.

Itolizumab (ALZUMAB®) is a humanized IgG1 mAb developed by Biocon.Itolizumab completed successful Phase III studies in patients withmoderate to severe psoriasis. Itolizumab has received marketing approvalin India; an application for FDA approval has not been submitted.

Obinutuzumab (GAZYVA®), originally developed by Roche and being furtherdeveloped under a collaboration agreement with Biogen is a humanizedanti-CD20 mAb approved for treatment of chronic lymphocytic leukemia. Itis also being investigated in Phase III clinical trials for patientswith various lymphomas. Dosages of about 1,000 mg are being administeredvia IV infusion.

Certolizumab pegol (CIMZIA®) is a recombinant, humanized antibody Fab′fragment, with specificity for human tumor necrosis factor alpha (TNFα),conjugated to an approximately 40 kDa polyethylene glycol (PEG2MAL40K).The molecular weight of certolizumab pegol is approximately 91 kDa.

Other antibody therapeutics that can be formulated withviscosity-lowering organophosphates include CT-P6 from Celltrion, Inc.(Celltrion).

Antibody Therapeutics in Late-Stage Trials and Development

The progression of antibody therapeutics to late-stage clinicaldevelopment and regulatory review are proceeding at a rapid pace. In2014, there are more than 300 mAbs in clinical trials and 30commercially-sponsored antibody therapeutics undergoing evaluation inlate-stage studies. First marketing applications for two mAbs(vedolizumab and ramucirumab) were recently submitted to the FDA. Amgenis currently sponsoring multiple ongoing Phase III trials on the use ofbrodalumab in patients with plaque psoriasis, with additional trialsplanned or recruiting patients. XBiotech, Inc. has sponsored two Phase Iclinical trials of MABp1 (Xilonix) for patients with advanced cancer ortype-2 diabetes. Additional trials of MABp1 are recruiting patients.Multiple trials are sponsored by Medlmmune, LLC (“Medlmmune”) andunderway or recruiting patients for the treatment of leukemia withmoxetumomab pasudotox. Long-term safety and efficacy studies areunderway for the use of tildrakizumab for the treatment of chronicplaque psoriasis. Multiple phase II trials have recently completed forthe use of rilotumumab for the treatment of various cancers.

At least 28 mAbs are high-molecular-weight proteins currently in orhaving recently completed Phase III studies for the treatment ofinflammatory or immunological disorders, cancers, high cholesterol,osteoporosis, Alzheimer's disease, and infectious diseases. The mAbs inor having recently completed Phase III trials include AMG 145,elotuzumab, epratuzumab, farletuzumab (MORAb-003), gantenerumab(RG1450), gevokizumab, inotuzumab ozogamicin, itolizumab, ixekizumab,lebrikizumab, mepolizumab, naptumomab estafenatox, necitumumab,nivolumab, ocrelizumab, onartuzumab, racotumomab, ramucirumab,reslizumab, romosozumab, sarilumab, secukinumab, sirukumab, solanezumab,tabalumab, and vedolizumab. A mAb mixture (actoxumab and bezlotoxumab)is also being evaluated in Phase III trials. See, e.g., Reichert, MAbs5:1-4, 2013.

Vedolizumab is a mAb being developed by Millennium Pharmaceuticals, Inc(“Millennium”; a subsidiary of Takeda Pharmaceuticals Company, Ltd.(“Takeda”)). Vedolizumab was found safe and highly effective forinducing and maintaining clinical remission in patients with moderate tosevere ulcerative colitis. Phase III clinical trials showed it to meetthe objectives of inducing a clinical response and maintaining remissionin Crohn's and ulcerative colitis patients. Studies evaluating long-termclinical outcomes show close to 60% of patients achieving clinicalremission. A common dose of vedolizumab are 6 mg/kg by IV infusion.

Ramucirumab is a human mAb being developed for the treatment of solidtumors. Phase III clinical trials are ongoing for the treatment ofbreast cancer, metastatic gastric adenocarcinoma, non-small cell lungcancer, and other types of cancer. Ramucirumab, in some Phase IIItrials, is administered at about 8 mg/kg via IV infusion.

Rilotumumab is a human mAb that inhibits the action of hepatocyte growthfactor/scatter factor. Developed by Amgen, it is in Phase III trials asa treatment for solid tumors. An open Phase III study of rilotumumabtreatment in patients with advanced or metastatic esophageal cancer willadminister rilotumumab at about 15 mg/kg via IV infusion.

Evolocumab (AMG 145), also developed by Amgen, is a mAb that binds toPCSK9. Evolocumab is indicated for hypercholesterolemia andhyperlipidemia.

Alirocumab (REGN727) is a human mAb from Regeneron Pharmaceuticals, Inc.(“Regeneron”) and Sanofi-Aventis U.S. LLC (“Sanofi”), indicated forhypercholesterolemia and acute coronary syndrome.

Naptumomab estafenatox, ABR-217620 from Active Biotech AB (“ActiveBiotech”) is a mAb indicated for renal cell carcinoma.

Racotumomab from CIMAB, SA (“CIMAB”); Laboratorio Elea S.A.C.I.F.y A. isa mAb indicated for non-small cell lung cancer.

Other antibodies which may be formulated with viscosity-loweringorganophosphates include bococizumab (PF-04950615) and tanezumab;ganitumab, blinatumomab, trebananib from Amgen; Anthrax immune globulinfrom Cangene Corporation; teplizumab from MacroGenics, Inc.; MK-3222,MK-6072 from Merck & Co (“Merck”); girentuximab from Wilex AG; RIGScanfrom Navidea Biopharmaceuticals (“Navidea”); PF-05280014 from Pfizer;SA237 from Chugai Pharmaceutical Co. Ltd. (“Chugai”); guselkumab fromJanssen/Johnson and Johnson Services, Inc. (“J&J”); Antithrombin Gamma(KW-3357) from Kyowa; and CT-P10 from Celltrion.

Antibodies in Early-Stage Clinical Trials

Many mAbs have recently entered, or are entering, clinical trials. Theycan include proteins currently administered via IV infusion, preferablythose having a molecular weight greater than about 120 kDa, typicallyfrom about 140 kDa to about 180 kDa. They can also include suchhigh-molecular-weight proteins such as Albumin-conjugated drugs orpeptides that are also entering clinical trials or have been approved bythe FDA. Many mAbs from Amgen are currently in clinical trials. Thesecan be high-molecular-weight proteins, for example, AMG 557, which is ahuman monoclonal antibody developed jointly by Amgen and AstraZeneca andcurrently in Phase I trials for treatment of lupus. Likewise, AMG 729 isa humanized mAb developed by Amgen and currently in Phase I trials forthe treatment of lupus and rheumatoid arthritis. In addition, AMG 110 isa mAb for epithelial cell adhesion molecule; AMG 157, jointly developedby Amgen and AstraZeneca, is a human mAb currently in Phase I for thetreatment of asthma; AMG 167 is a humanized mAb that has been evaluatedin multiple Phase I trials for the treatment of osteopenia; AMG 334,having completed Phase I dosing studies and currently in in Phase IIstudies for the treatment of migraines and hot flashes, is a human mAbthat inhibits Calcitonin Gene-Related Peptide; AMG 780 is a humananti-angiopoietin mAb that inhibits the interaction between theendothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2,and recently completed Phase I trials as a cancer treatment; AMG 811 isa human monoclonal antibody that inhibits interferon gamma beinginvestigated as a treatment for systemic lupus erythematosus; AMG 820 isa human mAb that inhibits c-fms and decreases tumor associatedmacrophage (TAM) function and is being investigated as a cancertreatment; AMG 181, jointly developed by Amgen and AstraZeneca, is ahuman mAb that inhibits the action of alpha4/beta7 and is in Phase IItrials as a treatment for ulcerative colitis and Crohn's disease.

Many mAbs are currently in clinical trials for the treatment ofautoimmune disorders. These mAbs can be included in low-viscosity,high-molecular-weight liquid formulations. RG7624 is a fully human mAbdesigned to specifically and selectively bind to the humaninterleukin-17 family of cytokines. A Phase I clinical trial evaluatingRG7624 for autoimmune disease is ongoing. BIIB033 is an anti-LINGO-1 mAbby Biogen currently in Phase II trials for treating multiple sclerosis.

High-molecular-weight proteins also can include AGS-009, a mAb targetingIFN-alpha developed by Argos Therapeutics, Inc. that recently completedphase I trials for the treatment of lupus. Patients are administered upto 30 mg/kg of AGS-009 via IV infusion. BT-061, developed by AbbVie, isin Phase II trials for patients with rheumatoid arthritis. Certolizumabpegol (CIMZIA®) is a mAb in Phase II trials for ankylosing spondylitisand juvenile rheumatoid arthritis. Clazakizumab, an anti-IL6 mAb, is inPhase II trials by Bristol-Myers Squibb.

CNTO-136 (sirukumab) and CNTO-1959 are mABs having recently completedPhase II and Phase III trials by Janssen. Daclizumab (previouslymarketed as ZENAPAX® by Roche) is currently in or has recently completedmultiple Phase III trials by AbbVie for the treatment of multiplesclerosis. Epratuzumab is a humanized mAb in Phase III trials for thetreatment of lupus. Canakinumab (MARIS®) is a human mAb targeted atinterleukin-1 beta. It was approved for the treatment ofcryopyrin-associated periodic syndromes. Canakinumab is in Phase Itrials as a possible treatment for chronic obstructive pulmonarydisease, gout and coronary artery disease. Mavrilimumab is a human mAbdesigned for the treatment of rheumatoid arthritis. Discovered asCAM-3001 by Cambridge Antibody Technology, mavrilimumab is beingdeveloped by MedImmune.

MEDI-546 are MEDI-570 are mAbs currently in Phase I and Phase II trialsby AstraZeneca for the treatment of lupus. MEDI-546 is administered inthe Phase II study by regular IV infusions of 300-1,000 mg. MEDI-551,another mAb being developed by AstraZeneca for numerous indications, isalso currently administered by IV infusion. NN8209, a mAb blocking theC5aR receptor being developed by Novo Nordisk A/S(“Novo Nordisk”), hascompleted a Phase II dosing study for treatment of rheumatoid arthritis.NN8210 is another antiC5aR mAb being developed by Novo Nordisk andcurrently is in Phase I trials. IPH2201 (NN8765) is a humanized mAbtargeting NKG2A being developed by Novo Nordisk to treat patients withinflammatory conditions and autoimmune diseases. NN8765 recentlycompleted Phase I trials.

Olokizumab is a humanized mAb that potently targets the cytokine IL-6.IL-6 is involved in several autoimmune and inflammatory pathways.Olokizumab has completed Phase II trials for the treatment of rheumatoidarthritis. Otelixizumab, also known as TRX4, is a mAb, which is beingdeveloped for the treatment of type 1 diabetes, rheumatoid arthritis,and other autoimmune diseases. Ozoralizumab is a humanized mAb that hascompleted Phase II trials.

Pfizer currently has Phase I trials for the mAbs PD-360324 andPF-04236921 for the treatment of lupus. A rituximab biosimilar,PF-05280586, has been developed by Pfizer and is in Phase I/Phase IItrials for rheumatoid arthritis.

Rontalizumab is a humanized mAb being developed by Genentech. Itrecently completed Phase II trials for the treatment of lupus. SAR113244(anti-CXCR5) is a mAb by Sanofi in Phase I trials. Sifalimumab(anti-IFN-alpha mAb) is a mAb in Phase II trials for the treatment oflupus.

A high-molecular-weight low-viscosity liquid formulation can include oneof the mAbs in early stage clinical development for treating variousblood disorders. For example, Belimumab (BENLYSTA®) has recentlycompleted Phase I trials for patients with vasculitis. Other mAbs inearly-stage trials for blood disorders include BI-655075 from BoehringerIngelheim GmbH “Boehringer Ingelheim”, ferroportin mAb and hepcidin mAbfrom Eli Lily, and Se1G1 from Selexys Pharmaceuticals, Corp.(“Selexys”).

One or more mAbs in early-stage development for treating various cancersand related conditions can be included in a low-viscosity,high-molecular-weight liquid formulation. United Therapeutics,Corporation has two mAbs in Phase I trials, 8H9 mAb and ch14.18 mAb. ThemAbs ABT-806, enavatuzumab, and volociximab from AbbVie are inearly-stage development. Actinium Pharmaceuticals, Inc has conductedearly-stage trials for the mAbs Actimab-A (M195 mAb), anti-CD45 mAb, andIomab-B. Seattle Genetics, Inc. (“Seattle Genetics”) has several mAbs inearly-stage trials for cancer and related conditions, includinganti-CD22 ADC (RG7593; pinatuzumab vedotin), anti-CD79b ADC (RG7596),anti-STEAP1 ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc.(“Agensys”) the antibody-drug conjugate RG7458, and vorsetuzumabmafodotin. The early-stage cancer therapeutics from Genentech can beincluded in low-viscosity formulations, including ALT-836, theantibody-drug conjugates RG7600 and DEDN6526A, anti-CD22 ADC (RG7593),anti-EGFL7 mAb (RG7414), anti-HER3/EGFR DAF mAb (RG7597), anti-PD-L1 mAb(RG7446), DFRF4539A, an MINT1526A. Bristol-Myers Squibb is developingearly-stage mAbs for cancer therapeutics, including those identified asanti-CXCR4, anti-PD-L1, IL-21 (BMS-982470), lirilumab, and urelumab(anti-CD137). Other mAbs in early-stage trials as cancer therapeuticsinclude APN301(hu14.18-IL2) from Apeiron Biologics AG, AV-203 from AVEOPharmaceuticals, Inc. (“AVEO”), AVX701 and AVX901 from AlphaVax, BAX-69from Baxter International, Inc. (“Baxter”), BAY 79-4620 and BAY 20-10112from Bayer HealthCare AG, BHQ880 from Novartis AG,212-Pb-TCMCtrastuzumab from AREVA Med, AbGn-7 from AbGenomicsInternational Inc, and ABIO-0501 (TALL-104) from Abiogen Pharma S.p.A.

Other antibody therapeutics that can be formulated withviscosity-lowering organophosphates include alzumab, GA101, daratumumab,siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumab (BYM338), CT-011(pidilizumab), actoxumab/bezlotoxumab (MK-3515A), MK-3475(pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212),AMG 139 (MEDI2070), SAR339658, dupilumab (REGN668), SAR156597,SAR256212, SAR279356, SAR3419, SAR153192 (REGN421, enoticumab),SAR307746 (nesvacumab), SAR650984, SAR566658, SAR391786, SAR228810,SAR252067, SGN-CD19A, SGN-CD33A, SGN-LIV1A, ASG 15ME, Anti-LINGO,BIIB037, ALXN1007, teprotumumab, concizumab, anrukinzumab (IMA-638),ponezumab (PF-04360365), PF-03446962, PF-06252616, etrolizumab (RG7413),quilizumab, ranibizumab, lampalizumab, onclacumab, gentenerumab,crenezumab (RG7412), IMC-RON8 (narnatumab), tremelimumab, vantictumab,eemcizumab, ozanezumab, mapatumumab, tralokinumab, XmAb5871, XmAb7195,cixutumumab (LY3012217), LY2541546 (blosozumab), olaratumab (LY3012207),MEDI4893, MEDI573, MEDI0639, MEDI3617, MEDI4736, MEDI6469, MEDI0680,MEDI5872, PF-05236812 (AAB-003), PF-05082566, BI 1034020, RG7116,RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652 (MPSK3169A),RG7686, HuMaxTFADC, MOR103, BT061, MOR208, OMP59R5 (anti-notch 2/3),VAY736, MOR202, BAY94-9343, LJM716, OMP52M51, GSK933776, GSK249320,GSK1070806, NN8828, CEP-37250/KHK2804 AGS-16M8F, AGS-16C3F, LY3016859,LY2495655, LY2875358, and LY2812176.

Other early stage mAbs that can be formulated with viscosity-loweringorganophosphates include benralizumab, MEDI-8968, anifrolumab, MEDI7183,sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune;BAN2401 from Biogen Idec/Eisai Co. LTD (“Eisai”)/BioArctic NeuroscienceAB; CDP7657 an anti-CD40L monovalent pegylated Fab antibody fragment,STX-100 an anti-avB6 mAb, BIIB059, Anti-TWEAK (BIIB023), and BIIB022from Biogen; fulranumab from Janssen and Amgen; BI-204/RG7418 fromBioInvent International/Genentech; BT-062 (indatuximab ravtansine) fromBiotest Pharmaceuticals Corporation; XmAb from BoehringerIngelheim/Xencor; anti-IP10 from Bristol-Myers Squibb; J 591 Lu-177 fromBZL Biologics LLC; CDX-011 (glembatumumab vedotin), CDX-0401 fromCelldex Therapeutics; foravirumab from Crucell; tigatuzumab from DaiichiSankyo Company Limited; MORAb-004, MORAb-009 (amatuximab) from Eisai;LY2382770 from Eli Lilly; DI17E6 from EMD Serono Inc.; zanolimumab fromEmergent BioSolutions, Inc.; FG-3019 from FibroGen, Inc.; catumaxomabfrom Fresenius SE & Co. KGaA; pateclizumab, rontalizumab from Genentech;fresolimumab from Genzyme & Sanofi; GS-6624 (simtuzumab) from Gilead;CNTO-328, bapineuzumab (AAB-001), carlumab, CNTO-136 from Janssen; KB003from KaloBios Pharmaceuticals, Inc.; ASKP1240 from Kyowa; RN-307 fromLabrys Biologics Inc.; ecromeximab from Life Science Pharmaceuticals;LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-HCV1 fromMassBiologics; AME-133v from MENTRIK Biotech, LLC; abituzumab from MerckKGaA; MM-121 from Merrimack Pharmaceuticals, Inc.; MCS110, QAX576,QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMACorporation (“XOMA”); NN8555 from Novo Nordisk; bavituximab, cotara fromPeregrine Pharmaceuticals, Inc.; PSMA-ADC from ProgenicsPharmaceuticals, Inc.; oregovomab from Quest Pharmatech, Inc.; fasinumab(REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331,RG7745 from Roche; ibalizumab (TMB-355) from TaiMed Biologics Inc.;TCN-032 from Theraclone Sciences; TRC105 from TRACON Pharmaceuticals,Inc.; UB-421 from United Biomedical Inc.; VB4-845 from Viventia Bio,Inc.; ABT-110 from AbbVie; Caplacizumab, Ozoralizumab from Ablynx; PRO140 from CytoDyn, Inc.; GS-CDA1, MDX-1388 from Medarex, Inc.; AMG 827,AMG 888 from Amgen; ublituximab from TG Therapeutics Inc.; TOL101 fromTolera Therapeutics, Inc.; huN901-DM1 (lorvotuzumab mertansine) fromImmunoGen Inc.; epratuzumab Y-90/veltuzumab combination (IMMU-102) fromImmunomedics, Inc.; anti-fibrin mAb/3B6/22 Tc-99m from Agenix, Limited;ALD403 from Alder Biopharmaceuticals, Inc.; RN6G/PF-04382923 fromPfizer; CG201 from CG Therapeutics, Inc.; KB001-A from KaloBiosPharmaceuticals/Sanofi; KRN-23 from Kyowa.; Y-90 hPAM 4 fromImmunomedics, Inc.; Tarextumab from Morphosys AG & OncoMedPharmaceuticals, Inc.; LFG316 from Morphosys AG & Novartis AG; CNTO3157,CNT06785 from Morphosys AG & Jannsen; RG6013 from Roche & Chugai; MM-111from Merrimack Pharmaceuticals, Inc. (“Merrimack”); GSK2862277 fromGlaxoSmithKline; AMG 282, AMG 172, AMG 595, AMG 745, AMG 761 from Amgen;BVX-20 from Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 fromCelltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776Afrom GlaxoSmithKline; anetumab ravtansine from Morphosys AG & Bayer AG;BI-836845 from Morphosys AG & Boehringer Ingelheim; NOV-7, NOV-8 fromMorphosys AG & Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 fromMerrimack, RG7882 from Roche & Seattle Genetics; RG7841 fromRoche/Genentech; PF-06410293, PF-06438179, PF-06439535, PF-04605412,PF-05280586 from Pfizer; RG7716, RG7936, gentenerumab, RG7444 fromRoche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212,MEDI-5117, MEDI-7814 from Astrazeneca; ulocuplumab, PCSK9 adnectin fromBristol-Myers Squibb; FPA009, FPA145 from FivePrime Therapeutics, Inc.;GS-5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin;MM-141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500,REGN1908-1909, REGN2009, REGN2176-3, REGN728 from Regeneron; SAR307746from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141, ALX-0171 fromAblynx; milatuzumab-DOX, milatuzumab, TF2, from Immunomedics, Inc.;MLN0264 from Millennium; ABT-981from AbbVie; AbGn-168H from AbGenomicsInternational Inc.; ficlatuzumab from AVEO; BI-505 from BioInventInternational; CDX-1127, CDX-301 from Celldex Therapeutics; CLT-008 fromCellerant Therapeutics Inc.; VGX-100 from Circadian; U3-1565 fromDaiichi Sankyo Company Limited; DKN-01 from Dekkun Corp.; flanvotumab(TYRP1 protein), IL-1 ß antibody, IMC-CS4 from Eli Lilly; VEGFR3 mAb,IMC-TR1 (LY3022859) from Eli Lilly and ImClone, LLC; Anthim from ElusysTherapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 fromImmunoGen Inc.; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken;KB004 from KaloBios Pharmaceuticals; MGA271, MGAH22 from MacroGenics,Inc.; XmAb5574 from MorphoSys AG/Xencor; ensituximab (NPC-1C) fromNeogenix Oncology, Inc.; LFA102 from Novartis AG and XOMA; ATI355 fromNovartis AG; SAN-300 from Santarus Inc.; Se1G1 from Selexys; HuM195/rGelfrom Targa Therapeutics, Corp.; VX15 from Teva Pharmaceuticals,Industries Ltd. (“Teva”) and Vaccinex Inc.; TCN-202 from TheracloneSciences; XmAb2513, XmAb5872 from Xencor; XOMA 3AB from XOMA andNational Institute for Allergy and Infectious Diseases; neuroblastomaantibody vaccine from MabVax Therapeutics; Cytolin from CytoDyn, Inc.;Thravixa from Emergent BioSolutions Inc.; and FB 301 from CytovanceBiologics; rabies mAb from Janssen and Sanofi; flu mAb from Janssen andpartly funded by National Institutes of Health; MB-003 and ZMapp fromMapp Biopharmaceutical, Inc.; and ZMAb from Defyrus Inc.

Other Protein Therapeutics

The protein can be an enzyme, a fusion protein, a stealth or pegylatedprotein, vaccine or otherwise a biologically active protein (or proteinmixture). The term “enzyme,” as used herein, refers to the protein orfunctional fragment thereof that catalyzes a biochemical transformationof a target molecule to a desired product.

Enzymes as drugs have at least two important features, namely i) oftenbind and act on their targets with high affinity and specificity, andii) are catalytic and convert multiple target molecules to the desiredproducts. In certain embodiments, the protein can be PEGylated, asdefined herein.

The term “fusion protein,” as used herein, refers to a protein that iscreated from two different genes encoding for two separate proteins.Fusion proteins are generally produced through recombinant DNAtechniques known to those skilled in the art. Two proteins (or proteinfragments) are fused together covalently and exhibit properties fromboth parent proteins.

There are a number of fusion proteins that are on the market.

ENBREL® (Etanercept), is a fusion protein marketed by Amgen thatcompetitively inhibits TNF.

ELOCTATE®, Antihemophilic Factor (Recombinant), Fc Fusion Protein, is arecombinant DNA derived, antihemophilic factor indicated in adults andchildren with Hemophilia A (congenital Factor VIII deficiency) forcontrol and prevention of bleeding episodes, perioperative management,routine prophylaxis to prevent or reduce the frequency of bleedingepisodes.

EYLEA® (aflibercept) is a recombinant fusion protein consisting ofportions of human VEGF receptors 1 and 2 extracellular domains fused tothe Fc portion of human IgG1 formulated as an iso-osmotic solution forintravitreal administration. EYLEA (aflibercept) is a recombinant fusionprotein consisting of portions of human VEGF receptors 1 and 2extracellular domains fused to the Fc portion of human IgG1 formulatedas an iso-osmotic solution for intravitreal administration. Afliberceptis a dimeric glycoprotein with a protein molecular weight of 97kilodaltons (kDa) and contains glycosylation, constituting an additional15% of the total molecular mass, resulting in a total molecular weightof 115 kDa. Aflibercept is produced in recombinant Chinese hamster ovary(CHO) cells, marketed by Regeneron.

ALPROLIX™, Coagulation Factor IX (Recombinant), Fc Fusion Protein, is arecombinant DNA derived, coagulation Factor IX concentrate is indicatedin adults and children with hemophilia B for control and prevention ofbleeding episodes, perioperative management, routine prophylaxis toprevent or reduce the frequency of bleeding episodes.

Pegloticase (KRYSTEXXA®) is a drug for the treatment of severe,treatment-refractory, chronic gout, developed by SavientPharmaceuticals, Inc. and is the first drug approved for thisindication. Pegloticase is a pegylated recombinant porcine-like uricasewith a molecular weight of about 497 kDa. Pegloticase is currentlyadministered by IV infusions of about 8 mg/kg. High-molecular-weight,low-viscosity liquid formulations can include pegloticase, preferably ina concentration of about 300 mg/mL to about 800 mg/mL.

Alteplase (ACTIVASE®) is a tissue plasminogen activator produced byrecombinant DNA technology. It is a purified glycoprotein comprising 527amino acids and synthesized using the complementary DNA (cDNA) fornatural human tissue-type plasminogen activator obtained from a humanmelanoma cell line. Alteplase is administered via IV infusion of about100 mg immediately following symptoms of a stroke. In some embodiments,low-viscosity formulations are provided containing alteplase, preferablyin a concentration of about 100 mg/mL.

Glucarpidase (VORAXAZE®) is a FDA-approved drug for the treatment ofelevated levels of methotrexate (defined as at least 1 micromol/L)during treatment of cancer patients who have impaired kidney function.Glucarpidase is administered via IV in a single dose of about 50 IU/kg.In some embodiments, low-viscosity formulations are provided containingglucarpidase.

Alglucosidase alfa (LUMIZYME®) is an enzyme replacement therapy orphandrug for treatment of Pompe disease (glycogen storage disease type II),a rare lysosomal storage disorder. It has a molecular weight of about106 kDa and is currently administered by IV infusions of about 20 mg/kg.In some embodiments, a low-viscosity pharmaceutical formulation ofalglucosidase alfa is provided, preferably with a concentration of about100 mg/mL to about 2,000 mg/mL.

Pegdamase bovine (ADAGEN®) is a modified enzyme used for enzymereplacement therapy for the treatment of severe combinedimmunodeficiency disease (SCID) associated with a deficiency ofadenosine deaminase. Pegdamase bovine is a conjugate of numerous strandsof monomethoxypolyethylene glycol (PEG), molecular weight 5,000 Da,covalently attached to adenosine deaminase enzyme that has been derivedfrom bovine intestine.

α-Galactosidase is a lysosomal enzyme that catalyses the hydrolysis ofthe glycolipid, globotriaosylceramide (GL-3), to galactose and ceramidedihexoside. Fabry disease is a rare inheritable lysosomal storagedisease characterized by subnormal enzymatic activity of α-Galactosidaseand resultant accumulation of GL-3. Agalsidase alfa (REPLAGAL®) is ahuman α-galactosidase A enzyme produced by a human cell line. Agalsidasebeta (FABRAZYME®) is a recombinant human α-galactosidase expressed in aCHO cell line. Replagal is administered at a dose of 0.2 mg/kg everyother week by intravenous infusion for the treatment of Fabry diseaseand, off label, for the treatment of Gaucher disease. FABRAZYME® isadministered at a dose of 1.0 mg/kg body weight every other week by IVinfusion. Other lysosomal enzymes can also be used. For example, theprotein can be a lysosomal enzyme as described in US 2012/0148556.

Rasburicase (ELITEK®) is a recombinant urate-oxidase indicated forinitial management of plasma uric acid levels in pediatric and adultpatients with leukemia, lymphoma, and solid tumor malignancies who arereceiving anti-cancer therapy expected to result in tumor lysis andsubsequent elevation of plasma uric acid. ELITEK® is administered bydaily IV infusion at a dosage of 0.2 mg/kg.

Imiglucerase (CEREZYME®) is a recombinant analogue of human(3-glucocerebrosidase. Initial dosages range from 2.5 U/kg body weight 3times a week to 60 U/kg once every 2 weeks. CEREZYME® is administered byIV infusion.

Abraxane, paclitaxel-conjugated albumin, is approved for metastaticbreast cancer, non-small cell lung cancer, and late stage pancreaticcancer.

Taliglucerase alfa (ELEYSO®) is a hydrolytic lysosomalglucocerebroside-specific enzyme indicated for long-term enzymereplacement therapy for Type 1 Gaucher disease. The recommended dose is60 U/kg of body weight administered once every 2 weeks via intravenousinfusion.

Laronidase (ALDURAZYME®) is a polymorphic variant of the human enzymeα-L-iduronidase that is produced via CHO cell line. The recommendeddosage regimen of ALDURAZYME® is 0.58 mg/kg administered once weekly asan intravenous infusion.

Elosufase alfa (VIMIZIM®) is a human N-acetylgalactosamine-6-sulfataseproduced by CHO cell line by BioMarin Pharmaceuticals Inc (“BioMarin”).It was approved by the FDA on Feb. 14, 2014 for the treatment ofMucopolysaccharidosis Type IVA. It is administered weekly viaintravenous infusion at a dosage of 2 mg/kg.

Other biologics which may be formulated with viscosity-loweringorganophosphates include asparaginase Erwinia chrysanthemi (ERWINAZE®),incobotulinumtoxin A (XEOMIN®), EPOGEN® (epoetin Alfa), PROCRIT®(epoetin Alfa), ARANESP® (darbepoetin alfa), ORENCIA® (abatacept),BATASERON® (interferon beta-1b), NAGLAZYME® (galsulfase); ELAPRASE®(Idursulfase); MYOZYME® (LUMIZYME®, algucosidase alfa); VPRIV®(velaglucerase), abobotulinumtoxin A (DYSPORT®); BAX-326, Octocog alfafrom Baxter; Syncria from GlaxoSmithKline; liprotamase from Eli Lilly;Xiaflex (collagenase clostridium histolyticum) from Auxilium andBioSpecifics Technologies Corp.; anakinra from Swedish Orphan BiovitrumAB; metreleptin from Bristol-Myers Squibb; Avonex, Plegridy (BIIB017)from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (darbepoetinalfa), AMG531 (romiplostim), KRN125 (pegfilgrastim), KW-0761(mogamulizumab) from Kyowa; IB1001 from Inspiration Biopharmaceuticals;Iprivask from Canyon Pharmaceuticals Group.

Protein Therapeutics in Development

Versartis, Inc.'s VRS-317 is a recombinant human growth hormone (hGH)fusion protein utilizing the XTEN half-life extension technology. Itaims to reduce the frequency of hGH injections necessary for patientswith hGH deficiency. VRS-317 has completed a Phase II study, comparingits efficacy to daily injections of non-derivatized hGH, with positiveresults. Phase III studies are planned.

Vibriolysin is a proteolytic enzyme secreted by the Gram-negative marinemicroorganism, Vibrio proteolyticus. This endoprotease has specificaffinity for the hydrophobic regions of proteins and is capable ofcleaving proteins adjacent to hydrophobic amino acids. Vibriolysin iscurrently being investigated by Biomarin for the cleaning and/ortreatment of burns. Vibriolysin formulations are described in patent WO02/092014.

PEG-PAL (PEGylated recombinant phenylalanine ammonia lyase or “PAL”) isan investigational enzyme substitution therapy for the treatment ofphenylketonuria (PKU), an inherited metabolic disease caused by adeficiency of the enzyme phenylalanine hydroxylase (PAH). PEG-PAL isbeing developed as a potential treatment for patients whose bloodphenylalanine (Phe) levels are not adequately controlled by KUVAN®.PEG-PAL is now in Phase 2 clinical development to treat patients who donot adequately respond to KUVAN®.

Other protein therapeutics which may be formulated withviscosity-lowering organophosphates include Alprolix/rFIXFc,Eloctate/rFVIIIFc, BMN-190; BMN-250; Lamazyme; Galazyme; ZA-011;Sebelipase alfa; SBC-103; and HGT-1110. Additionally, fusion-proteinscontaining the XTEN half-life extension technology including, but notlimited to: VRS-317 GH-XTEN; Factor VIla, Factor VIII, Factor IX;PF05280602, VRS-859; Exenatide-XTEN; AMX-256; GLP2-2G/XTEN; and AMX-179Folate-XTEN-DM1 can be formulated with viscosity-loweringorganophosphates.

Other late-stage protein therapeutics which can be formulated withviscosity-lowering organophosphates include CM-AT from CureMark LLC;NN7999, NN7088, Liraglutide (NN8022), NN9211, Semaglutide (NN9535) fromNovo Nordisk; AMG 386, Filgrastim from Amgen; CSL-654, Factor VIII fromCSL Behring; LA-EP2006 (pegfilgrastim biosimilar) from Novartis AG;Multikine (leukocyte interleukin) from CEL-SCI Corporation; LY2605541,Teriparatide (recombinant PTH 1-34) from Eli Lilly; NU-100 from NuronBiotech, Inc.; Calaspargase Pegol from Sigma-Tau Pharmaceuticals, Inc.;ADI-PEG-20 from Polaris Pharmaceuticals, Inc.; BMN-110, BMN-702 fromBioMarin; NGR-TNF from Molmed S.p.A.; recombinant human Cl esteraseinhibitor from Pharming Group/Santarus Inc.; Somatropin biosimilar fromLG Life Sciences LTD; Natpara from NPS Pharmaceuticals, Inc.; ART123from Asahi Kasei Corporation; BAX-111 from Baxter; OBI-1 fromInspiration Biopharmaceuticals; Wilate from Octapharma AG; Talactoferrinalfa from Agennix AG; Desmoteplase from Lundbeck; Cinryze from Shire;RG7421 and Roche and Exelixis, Inc.; Midostaurin (PKC412) from NovartisAG; Damoctocog alfa pegol, BAY 86-6150, BAY 94-9027 from Bayer AG;Peginterferon lambda-1a, Nulojix (Belatacept) from Bristol-Myers Squibb;Pergoveris, Corifollitropin alfa (MK-8962) from Merck KGaA; recombinantcoagulation Factor IX Fc fusion protein (rFIXFc; BIIB029) andrecombinant coagulation Factor VIII Fc fusion protein (rFVIIIFc;BIIB031) from Biogen; and Myalept from AstraZeneca.

Other early stage protein biologics which can be formulated withviscosity-lowering organophosphates include Alferon LDO from HemispherxBioPharma, Inc.; SL-401 from Stemline Therapeutics, Inc.; PRX-102 fromProtalix Biotherapeutics, Inc.; KTP-001 from Kaketsuken/Teijin PharmaLimited; Vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001(PF-05236806) from Janssen; LY2510924, LY2944876 from Eli Lilly; NN9924from Novo Nordisk; INGAP peptide from Exsulin; ABT-122 from Abbvie;AZD9412 from AstraZeneca; NEUBLASTIN (BG00010) from Biogen; Luspatercept(ACE-536), Sotatercept (ACE-011) from Celgene Corporation; PRAMEimmunotherapeutic from GlaxoSmithKline; Plovamer acetate (PI-2301) fromMerck KGaA; PREMIPLEX (607) from Shire; BMN-701 from BioMarin; Ontakfrom Eisai; rHuPH20/insulin from Halozyme, Inc.; PB-1023 from PhaseBioPharmaceuticals, Inc.; ALV-003 from Alvine Pharmaceuticals Inc. andAbbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon TherapeuticsInc.; PEGPH20 from Halozyme, Inc.; Amevive® alefacept from AstellasPharma Inc.; F-627 from Regeneron; AGN-214868 (senrebotase) fromAllergan, Inc.; BAX-817 from Baxter; PRT4445 from PortolaPharmaceuticals, Inc.; VEN100 from Ventria Bioscience;Onconase/ranpirnase from Tamir Biotechnology Inc.; interferon alpha-2binfusion from Medtronic,Inc; sebelipase alfa from Synageva BioPharma;IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline;SI-6603 from Seikagaku Corporation; ALXN1101, asfotase alfa fromAlexion; SHP611, SHP609 (Elaprase, idursulfase) from Shire; PF-04856884,PF-05280602 from Pfizer; ACE-031, Dalantercept from Acceleron Pharma;ALT-801 from Altor BioScience Corp.; BA-210 from BioAxone Biosciences,Inc.; WT1 immunotherapeutic from GlaxoSmithKline; GZ402666 from Sanofi;MSB0010445, Atacicept from Merck KGaA; Leukine (sargramostim) from BayerAG; KUR-211 from Baxter; fibroblast growth factor-1 from CardioVascularBioTherapeutics Inc.; SPI-2012 from Hanmi Pharmaceuticals Co.,LTD/Spectrum Pharmaceuticals; FGF-18 (sprifermin) from Merck KGaA;MK-1293 from Merck; interferon-alpha-2b from HanAll Biopharma; CYT107from Cytheris SA; RT001 from Revance Therapeutics, Inc.; MEDI6012 fromAztraZeneca; E2609 from Biogen; BMN-190, BMN-270 from BioMarin; ACE-661from Acceleron Pharma; AMG 876 from Amgen; GSK3052230 fromGlaxoSmithKline; RG7813 from Roche; SAR342434, Lantus from Sanofi; AZO1from Allozyne Inc.; ARX424 from Ambrx, Inc.; FP-1040, FP-1039 fromFivePrime Therapeutics, Inc.; ATX-MS-1467 from Merck KGaA; XTEN fusionproteins from Amunix Operating Inc.; entolimod (CBLB502) from ClevelandBioLabs, Inc.; HGT2310 from Shire; HM10760A from Hanmi PharmaceuticalsCo., LTD; ALXN1102/ALXN1103 from Alexion; CSL-689, CSL-627 from CSLBehring; glial growth factor 2 from Acorda Therapeutics, Inc.; NX001from Nephrx Corporation; NN8640, NN1436, NN1953, NN9926, NN9927, NN9928from Novo Nordisk; NHS-IL 12 from EMD Serono; 3K3A-APC from ZZ BiotechLLC; PB-1046 from PhaseBio Pharmaceuticals, Inc.; RU-101 from R-TechUeno, Ltd.; insulin lispro/BC106 from Adocia; hl-con1 from IconicTherapeutics, Inc.; PRT-105 from Protalix BioTherapeutics, Inc.;PF-04856883, CVX-096 from Pfizer; ACP-501 from AlphaCore Pharma LLC;BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-151 fromPromedior, Inc.; TS01 from Thrombolytic Science International; TT-173from Thrombotargets Corp.; QBI-139 from Quintessence Biosciences, Inc.;Vatelizumab, GBR500, GBR600, GBR830, and GBR900 from GlenmarkPharmaceuticals; and CYT-6091 from Cytimmune Sciences, Inc.

Other Biologic Agents

Other biologic drugs that can be formulated with viscosity-loweringorganophosphates include PF-05285401, PF-05231023, RN317 (PF-05335810),PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598,RG7842, RG7624d, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC-18F1,IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810(RN317) PD-0360324, PF-00547659 from Pfizer; MK-8237 from Merck; BI033from Biogen; GZ402665, SAR438584/REGN2222 from Sanofi; IMC-18F1; andIcrucumab, IMC-3G3 from ImClone LLC; Ryzodeg, Tresiba, Xultophy fromNovo Nordisk; Toujeo (U300), LixiLan, Lyxumia (lixisenatide) fromSanofi; MAGE-A3 immunotherapeutic from GlaxoSmithKline; Tecemotide fromMerck KGaA; Sereleaxin (RLX030) from Novartis AG; Erythropoietin;Pegfilgrastim; LY2963016, Dulaglutide (LY2182965) from Eli Lilly; andInsulin Glargine from Boehringer Ingelheim.

B. Organophosphates

The viscosity of liquid protein formulations, includinglow-molecular-weight and/or high-molecular-weight proteins, is reducedby the addition of one or more organophosphates. The pharmaceuticalformulations are in some cases converted from non-Newtonian to Newtonianfluids by the addition of an effective amount of one or moreorganophosphates. An “organophosphate” herein is a compound containingone or more phosphoryl groups at least one of which is covalentlyconnected to an organic group through a phosphoester bond. Theorganophosphate can be a monoester of a phosphoric acid orpolyphosphoric acid. The organophosphate can be a diester of aphosphoric acid or polyphosphoric acid. The organophosphate can be asalt or a zwitterion. The term “zwitterion” is used herein to describean overall neutrally charged chemical molecule which carries formalpositive and negative charges on different chemical groups in themolecule.

The organophosphate can be a salt or a zwitterion. The term “zwitterion”is used herein to describe an overall neutrally charged chemicalmolecule which carries formal positive and negative charges on differentchemical groups in the molecule.

When the organophosphate is in the form of a salt, the counterion may bean alkaline or alkaline earth metal, such as sodium, calcium, lithium,potassium and the like. In other embodiments, the counterion may be anitrogen-containing compound, including nitrogen containing compoundshaving sequential methylene and/or methine groups, benzene, naphthalene,camphor, adamantane, toluene, quinone, anthracene, phenanthrene,pyridine, pyrazine, piperazine, pyrrolidine, piperidine, imidazole,pyrazole, oxazole, thiophene, benzimidazole, or substituted analogsthereof. Exemplary nitrogen-containing compounds include, but are notlimited to, L-lysine, L-arginine, L-histidine, pentane-1,5- andhexane-1,6-diamine, adamantylamine,1-(3-aminopropyl)-2-methyl-1H-imidazole, aminomethylethyl pyrrolidine,dimethylaminopropylpiperazine, aminoethylpiperidine,aminoethylpiperazine, and ethanolamine. For example, the organophosphatecan be a salt of thiamine pyrophosphate and1-(3-aminopropyl)-2-methyl-1H-imidazole, referred to as TPP-APMI.

Although generally any organophosphate may lower the viscosity of aprotein formulation, in some embodiments the viscosity-reducingorganophosphate is a nucleotide or nucleotide derivative or contains anucleotide or nucleotide derivative. The viscosity-reducingorganophosphate can be a nucleotide monophosphate, a nucleotidediphosphate, a nucleotide triphosphate, or a derivative thereof. Theviscosity-reducing organophosphate can be a nucleoside monophosphate, anucleoside diphosphate, a nucleoside triphosphate, or a derivativethereof. The viscosity-reducing organophosphate can contain a nucleobaseor a derivative thereof. In some embodiments the viscosity-reducingorganophosphate is a conjugate of a nucleobase and a phosphoryl group; aconjugate of a sugar and a phosphoryl group; or a conjugate of anucleobase, a sugar, and a phosphoryl group. The sugar can be a 5-carbonsugar, a 6-carbon sugar, or a 7-carbon sugar, optionally having one ormore substituents. The nucleobase can be purine, adenine, guanine,hypoxanthine, xanthine, 7-methylguanine, pyrimidine, thymine, cytosine,uracil, 5, 6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine,or a derivative thereof. The nucleoside can be adenosine, guanosine,5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine,thymidine, deoxyuridine, deoxycytidine, or a derivative thereof. Thenucleotide can be a monophosphate, diphosphate, or triphosphate of anyof the nucleosides described above.

The viscosity-reducing organophosphate can have a structure according toFormula I wherein X is a phosphate, preferably a diphosphate ortriphosphate; Y is none or a sugar, preferably ribose, deoxyribose, or aderivative thereof; and Z is a nucleobase, preferably one of thosedescribed above or a derivative thereof.

X—Y—Z   Formula I

The viscosity-reducing organophosphate can have a structure according toFormula II wherein n is an integer from 1 to 20, from 1 to 10, from 2 to10, or from 2 to 6; wherein R¹ is an organic group having from 3 to 50carbon atoms, from 5 to 30 carbon atoms, or from 7 to 20 carbon atoms,preferably R¹ is a nucleobase, a nucleoside, or a derivative thereof,and wherein each occurrence of R² is independently selected from thegroup consisting of none, hydrogen, monovalent cationic groups, andorganic groups having from 1 to 50 carbon atoms, from 1 to 30 carbonatoms, from 3 to 30 carbon atoms, or from 7 to 20 carbon atoms.Monovalent cationic groups include potassium, sodium, lithium, ammonium,and alkyl ammonium groups. R¹ and R², whenever R² is an organic group,can be a substituted or unsubstituted carbocycle or heterocycle havingfrom 3 to 50 carbon atoms, from 5 to 30 carbon atoms, or from 7 to 20carbon atoms. R¹ can be a nucleoside such as one of those describedabove or a derivative thereof.

The viscosity-reducing organophosphate can have a structure according toFormula III wherein n is an integer from 1 to 20, from 1 to 10, from 2to 10, or from 2 to 6; wherein R³ is none or is a sugar, preferably amonosaccharide or disaccharide, having from 1 to 30 carbon atoms, from 1to 20 carbon atoms, or from 4 to 20 carbon atoms; wherein R⁴ is a bulkycyclic group that can be substituted or unsubstituted, preferably asubstituted or unsubstituted carbocycle or heterocycle having from 3 to50 carbon atoms, from 5 to 30 carbon atoms, or from 7 to 20 carbonatoms; and wherein each occurrence of R⁵ is independently selected fromthe group consisting of none, hydrogen, monovalent cationic groups, andorganic groups having from 1 to 50 carbon atoms, from 1 to 30 carbonatoms, from 3 to 30 carbon atoms, or from 7 to 20 carbon atoms. R³ canbe deoxyribose, fructose, galactose, gentiobiulose, gentiobiose,glucose, kestose, isomaltose, isomaltotriose, kojibiose, laminaribiose,maltose, maltulose, maltotriose, maltotriulose, mannobiose, mannose,melibiose, melibiulose, nigerose, nigerotriose, raffinose, ribose,rutinose, rutinulose, sophorose, trehalose, β,β-trehalose,α,β-trehalose, or turanose, optionally containing one or moresubstituents. In certain embodiments, the substituents R³ and R⁵ maytogether form a ring, for instance as found in cyclic adenosinemonophosphate.

R⁴ can be a nitrogen-containing heterocycle. Nitrogen-containingheterocycles can be saturated or unsaturated. Nitrogen-containingheterocycles can include substituted and unsubstituted pyrrolidine,pyrrole, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine,isoxazolidine, oxazole, isoxazole, piperidine, tetrahydropyridine,dihydropyridine, pyridine, pyrimidine, piperazine, polycyclic and fusedring structures thereof, and derivatives thereof. R⁴ can be a bulkycyclic group. Suitable bulky cyclic groups can include 5-memberedcarbocylces and heterocycles such as cyclopentane, cyclopentene,cyclopentadiene, pyrrolidine, pyrrole, imidazolidine, pyrazolidine,imidazole, pyrazole, oxazolidine, isoxazolidine, oxazole, isoxazole,tetrahydrofuran, furan, dioxolane, thiolane, thiophene, dithiolane,thiazole, isothiazole, phosphole, silole, triazole, oxadiazole, andderivatives thereof. Suitable bulky cyclic groups can include 6-memberedcarbocylces and heterocycles such as cyclohexane, cyclohexene,cyclohexa-1,3-diene, cyclohexa-1,4-diene, benzene, piperidine,tetrahydropyridine, dihydropyridine, pyridine, oxane, pyran, piperazine,pyrazine, pyrimidine, pyridazine, morpholine, 1,3,5-triazine, andderivatives thereof. Suitable bulky cyclic groups can include 7-memberedcarbocylces and heterocycles such as cycloheptane, cycloheptene,azepane, azepine, thiepine, diazepine, thiazepine, and derivativesthereof. Suitable bulky cyclic groups can include polycyclic compoundssuch as polycyclic and fused ring structures of any of the abovecarbocycles and heterocycles such as naphthalene, anthracene, tetracene,acridine, dibenzothiophene, carbazole, dibenzofuran, decalin, bridgedcarbocycles and heterocycles such as norbornane, adamantane, andspirocyclic compounds such as spiro[2.2]pentane.

The viscosity-reducing organophosphate can be a dinucleotide phosphate.The viscosity-reducing organophosphate can have a structure according toFormula IV wherein n is an integer from 1 to 20, from 1 to 10, from 2 to10, or from 2 to 6; wherein each occurrence of R⁶ is independentlyselected from none or a sugar, preferably a monosaccharide ordisaccharide, having from 1 to 30 carbon atoms, from 1 to 20 carbonatoms, or from 4 to 20 carbon atoms; wherein each occurrence of R⁷ isindependently a bulky cyclic group that can be substituted orunsubstituted, preferably a substituted or unsubstituted carbocycle orheterocycle having from 3 to 50 carbon atoms, from 5 to 30 carbon atoms,or from 7 to 20 carbon atoms; and wherein each occurrence of R⁸ isindependently selected from the group consisting of none, hydrogen,monovalent cationic groups, and organic groups having from 1 to 50carbon atoms, from 1 to 30 carbon atoms, from 3 to 30 carbon atoms, orfrom 7 to 20 carbon atoms. Each R⁶ can independently be deoxyribose,fructose, galactose, gentiobiulose, gentiobiose, glucose, kestose,isomaltose, isomaltotriose, kojibiose, laminaribiose, maltose,maltulose, maltotriose, maltotriulose, mannobiose, mannose, melibiose,melibiulose, nigerose, nigerotriose, raffinose, ribose, rutinose,rutinulose, sophorose, trehalose, β,β-trehalose, α,β-trehalose, orturanose, optionally containing one or more substituents. Each R⁷ canindependently be a nitrogen-containing heterocycle. Nitrogen-containingheterocycles can be saturated or unsaturated. Nitrogen-containingheterocycles can include substituted and unsubstituted pyrrolidine,pyrrole, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine,isoxazolidine, oxazole, isoxazole, piperidine, tetrahydropyridine,dihydropyridine, pyridine, pyrimidine, piperazine, polycyclic and fusedring structures thereof, and derivatives thereof. Each R⁷ canindependently be a bulky cyclic group. Suitable bulky cyclic groups caninclude 5-membered carbocylces and heterocycles such as cyclopentane,cyclopentene, cyclopentadiene, pyrrolidine, pyrrole, imidazolidine,pyrazolidine, imidazole, pyrazole, oxazolidine, isoxazolidine, oxazole,isoxazole, tetrahydrofuran, furan, dioxolane, thiolane, thiophene,dithiolane, thiazole, isothiazole, phosphole, silole, triazole,oxadiazole, and derivatives thereof. Suitable bulky cyclic groups caninclude 6-membered carbocylces and heterocycles such as cyclohexane,cyclohexene, cyclohexa-1,3-diene, cyclohexa-1,4-diene, benzene,piperidine, tetrahydropyridine, dihydropyridine, pyridine, oxane, pyran,piperazine, pyrazine, pyrimidine, pyridazine, morpholine,1,3,5-triazine, and derivatives thereof. Suitable bulky cyclic groupscan include 7-membered carbocylces and heterocycles such ascycloheptane, cycloheptene, azepane, azepine, thiepine, diazepine,thiazepine, and derivatives thereof. Suitable bulky cyclic groups caninclude polycyclic compounds such as polycyclic and fused ringstructures of any of the above carbocycles and heterocycles such asnaphthalene, anthracene, tetracene, acridine, dibenzothiophene,carbazole, dibenzofuran, decalin; bridged carbocycles and heterocyclessuch as norbornane, adamantane, and spirocyclic compounds such asspiro[2.2]pentane.

The viscosity-reducing organophosphate can be thiamine pyrophosphate(TPP), the structure of which is shown below as a chloride salt, or aderivative thereof. Derivatives of TPP can include replacing thediphosphate with a different phosphate such as monophosphate totriphosphate; replacing the chloride anion with other anionicconstituents; replacing one or more methyl substitutents withhigher-order alkyl or N-alkyl substituents; replacing one or more aminosubstituents with substituted or unsubstituted alkyl, aminoalkyl,heterocyclyl, aryl, or heteroaryl groups having from 1 to 30 carbonatoms; replacing one or more hydroxyl groups with O-acyl or O-alkylgroups; or a combination thereof. Suitable anionic constituents includehalide ions, sulfate, sulfonate, sulfite, sulfinate, phosphate,phosphonate, phosphite, phosphonite, carbonate, and carboxylate anionsoptionally substituted with one or more alkyl, heteroalkyl, alkenyl,alkynyl, carbocyclic, or heterocyclic groups, preferably having from 1to 20 or from 1 to 12 carbon atoms. Exemplary anionic constituentsinclude chloride, bromide, methylphosphate, methyl-ethyl-phosphate,methylsulfate, methylsulfonate, formate, acetate, butyrate, citrate, andlactate and bulky hydrophobic anions such as camphor sulfonic acid(CSA), benzene sulfonic acid (BSA), toluene sulfonic acid (TSA),1-(3-aminopropyl)-2-methyl-1H-imidazole (APMI), or methane sulfonic acid(MSA). Derivatives can include base addition salts of TPP using commoninorganic bases such as NaOH or exemplary hydrophobic bases describedabove.

In other embodiments, the viscosity-reducing organophosphate may bebenfotiamine or the corresponding diphosphate or triphosphate analog.The viscosity-reducing organophosphate may fursultiamine monophosphate,prosultiamine monophosphate, or allithiamine monophosphate, as well asthe corresponding diphosphate or triphosphate of any of the above.

The viscosity-reducing organophosphate can be adenosine triphosphate(ATP), the structure of which is shown below as a sodium salt, or aderivative thereof. Derivatives of ATP can include replacing thetriphosphate with a different phosphate such as monophosphate ordiphosphate; replacing the amino substituent with substituted orunsubstituted alkyl, aminoalkyl, aryl, heterocyclyl or heteroaryl groupshaving from 1 to 30 carbon atoms; replacing one or more hydroxyl groupswith O-acyl or O-alkyl groups; or a combination thereof.

The viscosity-reducing organophosphate can be deoxyadenosinetriphosphate (dATP), the structure of which is shown below, or aderivative thereof.

Derivatives of dATP can include replacing the triphosphate with adifferent phosphate such as monophosphate or diphosphate; replacing theamino substituent with substituted or unsubstituted alkyl, aminoalkyl,aryl, heterocyclyl, or heteroaryl groups having from 1 to 30 carbonatoms; replacing one or more hydroxyl groups with O-acyl or O-alkylgroups; or a combination thereof.

The viscosity-reducing organophosphate can be deoxyguanosinetriphosphate (dGTP), the structure of which is shown below, or aderivative thereof. Derivatives of dGTP can include replacing thetriphosphate with a different phosphate such as monophosphate ordiphosphate; replacing the amino substituent with substituted orunsubstituted alkyl, aminoalkyl, heterocyclyl, aryl, or heteroarylgroups having from 1 to 30 carbon atoms; replacing one or more hydroxylgroups with O-acyl or O-alkyl groups; or a combination thereof.

The viscosity-reducing organophosphate can be deoxythymidinetriphosphate (dTTP), the structure of which is shown below, or aderivative thereof. Derivatives of dTTP can include replacing thetriphosphate with a different phosphate such as monophosphate ordiphosphate; replacing the methyl substituent with higher-order alkyl orN-alkyl substituents; replacing one or more amino substituents withsubstituted or unsubstituted alkyl, aminoalkyl, heterocyclyl, aryl, orheteroaryl groups having from 1 to 30 carbon atoms; replacing one ormore hydroxyl groups with O-acyl or O-alkyl groups; or a combinationthereof

The viscosity-reducing organophosphate can be deoxycytidine triphosphate(dCTP), the structure of which is shown below, or a derivative thereof.Derivatives of dCTP can include replacing the triphosphate with adifferent phosphate such as monophosphate or diphosphate; replacing theamino substituent with substituted or unsubstituted alkyl, aminoalkyl,aryl, heterocyclyl, or heteroaryl groups having from 1 to 30 carbonatoms; replacing one or more hydroxyl groups with O-acyl or O-alkylgroups; or a combination thereof.

The viscosity-reducing organophosphate can be cyclic adenosinemonophosphate (cAMP), the structure of which is shown below, or aderivative thereof. Derivatives of cAMP can include replacing themonophosphate with a different phosphate such as diphosphate ortriphosphate; replacing the amino substituent with substituted orunsubstituted alkyl, aminoalkyl, aryl, heterocyclyl, or heteroarylgroups having from 1 to 30 carbon atoms; replacing the hydroxyl groupwith O-acyl or O-alkyl groups; or a combination thereof

The viscosity-reducing organophosphate can be cyclic guanosinemonoosphate (cGMP), the structure of which is shown below, or aderivative thereof. Derivatives of cGMP can include replacing themonophosphonate with a different phosphate such as diphosphate ortriphosphate; replacing the amino substituent with substituted orunsubstituted alkyl, aminoalkyl, aryl, heterocyclyl, or heteroarylgroups having from 1 to 30 carbon atoms; replacing one or more hydroxylgroups with O-acyl or O-alkyl groups; or a combination thereof.

The viscosity-reducing organophosphate can be cyclic thymidinemonophosphate (cTMP), the structure of which is shown below, or aderivative thereof. Derivatives of cTMP can include replacing themonophosphonate with a different phosphate such as diphosphate ortriphosphate; replacing the methyl substituent with higher-order alkylor N-alkyl substituents; replacing one or more amino substituents withsubstituted or unsubstituted alkyl, aminoalkyl, aryl, heterocyclyl, orheteroaryl groups having from 1 to 30 carbon atoms; replacing one ormore hydroxyl groups with O-acyl or O-alkyl groups; or a combinationthereof

The viscosity-reducing organophosphate can be cyclic cytidinemonophosphate (cCMP), the structure of which is shown below, or aderivative thereof. Derivatives of cCMP can include replacing themonophosphonate with a different phosphate such as diphosphate ortriphosphate; replacing the amino substituent with substituted orunsubstituted alkyl, aminoalkyl, aryl, heterocyclyl, or heteroarylgroups having from 1 to 30 carbon atoms; replacing one or more hydroxylgroups with O-acyl or O-alkyl groups; or a combination thereof.

The viscosity-reducing organophosphate can be nicotinamide adeninedinucleotide phosphate (NADP), the structure of which is shown below asa sodium salt, or a derivative thereof. Derivatives of NADP can includereplacing the diphosphate with a different phosphate such asmonophosphate or triphosphate; replacing the diphosphonate with adifferent phosphate; replacing one or more amino substituents withsubstituted or unsubstituted alkyl, aminoalkyl, heterocyclyl, aryl, orheteroaryl groups having from 1 to 30 carbon atoms; replacing one ormore hydroxyl groups with O-acyl or O-alkyl groups; or a combinationthereof

The viscosity-reducing organophosphate can be pyridoxal phosphate, thestructure of which is shown below, or a derivative thereof. Derivativesof pyridoxal phosphate can include replacing the monophosphate with adifferent phosphate such as diphosphate or triphosphate; replacing themethyl substituent with higher-order alkyl or N-alkyl substituents;replacing one or more hydroxyl groups with O-acyl or O-alkyl groups; ora combination thereof.

The viscosity-reducing organophosphate can be riboflavin-5′-phosphate,the structure of which is shown below, or a derivative thereof.Derivatives of riboflavin-5′-phosphate can include replacing thephosphate with a different phosphate such as a diphosphate ortriphosphate; replacing the sodium counter ion with other cationicconstituents; replacing one or more methyl substituents withhigher-order alkyl or N-alkyl substituents; replacing one or morehydroxyl groups with O-acyl or O-alkyl groups; or combinations thereof

C. Excipients

A wide variety of pharmaceutical excipients useful for liquid proteinformulations are known to those skilled in the art. They include one ormore additives, such as liquid solvents or co-solvents; sugars or sugaralcohols such as mannitol, trehalose, sucrose, sorbitol, fructose,maltose, lactose, or dextrans; surfactants such as TWEEN® 20, 60, or 80(polysorbate 20, 60, or 80); buffering agents; preservatives such asbenzalkonium chloride, benzethonium chloride, tertiary ammonium salts,and chlorhexidinediacetate; carriers such as poly(ethylene glycol)(PEG); antioxidants such as ascorbic acid, sodium metabisulfite, andmethionine; chelating agents such as EDTA or citric acid; orbiodegradable polymers such as water soluble polyesters;cryoprotectants; lyoprotectants; bulking agents; and stabilizing agents.

Other pharmaceutically acceptable carriers, excipients, or stabilizers,such as those described in Remington: “The Science and Practice ofPharmacy”, 20th edition, Alfonso R. Gennaro, Ed., Lippincott Williams &Wilkins (2000) may also be included in a protein formulation describedherein, provided that they do not adversely affect the desiredcharacteristics of the formulation.

The formulations, in addition to the viscosity-reducing organophosphatesdescribed above, can contain one or more additional viscosity-loweringexcipients. The organophosphate viscosity-lowering agents describedherein can be combined with one or more other types ofviscosity-lowering agents, for example, water soluble organic dyesdescribed in co-filed PCT application entitled LIQUID PROTEINFORMULATIONS CONTAINING WATER SOLUBLE ORGANIC DYES by ArsiaTherapeutics; the typically bulky polar organic compounds, such ashydrophobic compounds, many of the GRAS (US Food and Drug AdministrationList of compounds Generally Regarded As Safe) and inactive injectableingredients and FDA approved therapeutics, LIQUID PROTEIN

FORMULATIONS CONTAINING VISCOSITY-LOWERING AGENTS by Arsia Therapeutics;and ionic liquids described in co-filed PCT application entitled LIQUIDPROTEIN FORMULATIONS CONTAINING IONIC LIQUIDS by Arsia Therapeutics.

III. Methods of Making

A. Protein Preparation

The protein, such as a mAb, to be formulated may be produced by anyknown technique, such as by culturing cells transformed or transfectedwith a vector containing one or more nucleic acid sequences encoding theprotein, as is well known in the art, or through synthetic techniques(such as recombinant techniques and peptide synthesis or a combinationof these techniques), or may be isolated from an endogenous source ofthe protein.

Purification of the protein to be formulated may be conducted by anysuitable technique known in the art, such as, for example, ethanol orammonium sulfate precipitation, reverse phase HPLC, chromatography onsilica or cation-exchange resin (e.g., DEAE-cellulose), dialysis,chromatofocusing, gel filtration using protein A SEPHAROSE® columns(e.g., SEPHADEX® G-75) to remove contaminants, metal chelating columnsto bind epitope-tagged forms, and ultrafiltration/diafiltration(non-limiting examples include centrifugal filtration and tangentialflow filtration (TFF)).

Inclusion of viscosity-reducing organophosphates at viscosity-reducingconcentrations such as 0.010 M to 1.0 M, preferably 0.050 M to 0.50 M,most preferably 0.10 M to 0.30 M, allows a solution of thepharmaceutically active mAb to be purified and/or concentrated at highermAb concentrations using common methods known to those skilled in theart, including but not limited to tangential flow filtration,centrifugal concentration, and dialysis.

In some embodiments, lyophilized formulations of the proteins areprovided and/or are used in the preparation and manufacture of thelow-viscosity, concentrated protein formulations. In some embodiments,the pre-lyophilized protein in a powder form is reconstituted bydissolution in an aqueous solution. In this embodiment, the liquidformulation is filled into a specific dosage unit container such as avial or pre-filled mixing syringe, lyophilized, optionally withlyoprotectants, preservatives, antioxidants, and other typicalpharmaceutically acceptable excipients, then stored under sterilestorage conditions until shortly before use, at which time it isreconstituted with a defined volume of diluent, to bring the liquid tothe desired concentration and viscosity.

The formulations described herein may be stored by any suitable methodknown to one skilled in the art. Non-limiting examples of methods forpreparing the protein formulations for storage include freezing,lyophilizing, and spray drying the liquid protein formulation. In somecases, the lyophilized formulation is frozen for storage at subzerotemperatures, such as at about −80° C. or in liquid nitrogen. In somecases, a lyophilized or aqueous formulation is stored at 2-8° C.

Non-limiting examples of diluents useful for reconstituting alyophilized formulation prior to injection include sterile water,bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solution,dextrose solution, or aqueous solutions of salts and/or buffers. In somecases, the formulation is spray-dried and then stored.

IV. Administration to an Individual in Need Thereof

The protein formulations, including, but not limited to, reconstitutedformulations, are administered to a person in need thereof byintramuscular, intraperitoneal (i.e., into a body cavity),intracerobrospinal, or subcutaneous injection using an 18-32 gaugeneedle (optionally a thin-walled needle), in a volume of less than about5 mL, less that about 3 mL, preferably less than about 2 mL, morepreferably less than about 1 mL.

The appropriate dosage (“therapeutically effective amount”) of theprotein, such as a mAb, will depend on the condition to be treated, theseverity and course of the disease or condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered at one time in single or multiple injections,or over a series of treatments, as the sole treatment, or in conjunctionwith other drugs or therapies.

Dosage formulations are designed so that the injections cause nosignificant signs of irritation at the site of injection, for example,wherein the primary irritation index is less than 3 when evaluated usinga Draize scoring system. In an alternative embodiment, the injectionscause macroscopically similar levels of irritation when compared toinjections of equivalent volumes of saline solution. In anotherembodiment, the bioavailability of the protein is higher when comparedto the otherwise same formulation without the organophosphateadministered in the same way.

In a preferred embodiment, the formulation is injected to yieldincreased levels of the therapeutic protein. For example, the AUC valuemay be at least 10%, preferably at least 20%, larger than the same valuecomputed for the otherwise same formulation without theviscosity-reducing organophosphate(s) administered in the same way.

The viscosity-reducing organophosphate may also affect bioavailability.For example, the percent bioavailability of the protein may be at least1.1 times, preferably at least 1.2 times the percent bioavailability ofthe otherwise same formulation without the viscosity-reducingorganophosphate(s) administered in the same way.

The viscosity-reducing organophosphate may also affect thepharmacokinetics. For example, the C_(MAX) after SC or IM injection maybe at least 10%, preferably at least 20%, less than the C_(MAX) of anapproximately equivalent pharmaceutically effective intravenouslyadministered dose.

In some embodiments, the proteins are administered at a higher dosageand a lower frequency than the otherwise same formulations without theviscosity-reducing organophosphate(s).

The lower viscosity formulations require less injection force. Forexample, the injection force may be at least 10%, preferably at least20%, less than the injection force for the otherwise same formulationwithout the viscosity-reducing organophosphate(s) administered in thesame way. In one embodiment, the injection is administered with a 27gauge needle and the injection force is less than 30 N. The formulationscan be administered in most cases using a very small gauge needle, forexample, between 27 and 31 gauge, typically 27, 29 or 31 gauge.

The viscosity-reducing organophosphate may be used to prepare a dosageunit formulation suitable for reconstitution to make a liquidpharmaceutical formulation for subcutaneous or intramuscular injection.The dosage unit may contain a dry powder of one or more proteins; one ormore viscosity-reducing organophosphates; and other excipients. Theproteins are present in the dosage unit such that after reconstitutionin a pharmaceutically acceptable solvent, the resulting formulation hasa protein concentration from about 100 mg to about 2,000 mg per 1 mL(mg/mL). Such reconstituted formulations may have an absolute viscosityof from about 1 cP to about 50 cP at 25° C.

The low viscosity formulation can be provided as a solution or in adosage unit form where the protein is lyophilized in one vial, with orwithout the viscosity-reducing organophosphate and the other excipients,and the solvent, with or without the viscosity-reducing organophosphateand other excipients, is provided in a second vial. In this embodiment,the solvent is added to the protein shortly before or at the time ofinjection to insure uniform mixing and dissolution.

The viscosity-reducing organophosphate(s) are present in theformulations at concentrations that cause no significant signs oftoxicity and/or no irreversible signs of toxicity when administered viasubcutaneous, intramuscular, or other types of injection. As usedherein, “significant signs of toxicity” include intoxication, lethargy,behavioral modifications such as those that occur with damage to thecentral nervous system, infertility, signs of serious cardiotoxicitysuch as cardiac arrhythmia, cardiomyopathy, myocardial infarctions, andcardiac or congestive heart failure, kidney failure, liver failure,difficulty breathing, and death.

In preferred embodiments the formulations cause no significantirritation when administered not more than twice daily, once daily,twice weekly, once weekly or once monthly. The protein formulations canbe administered causing no significant signs of irritation at the siteof injection, e.g. a primary irritation index of less than 3, less than2, or less than 1 when evaluated using a Draize scoring system. As usedherein, “significant signs of irritation” include erythema, redness,and/or swelling at the site of injection having a diameter of greaterthan 10 cm, greater than 5 cm, or greater than 2.5 cm, necrosis at thesite of injection, exfoliative dermatitis at the site of injection, andsevere pain that prevents daily activity and/or requires medicalattention or hospitalization. In some embodiments, injections of theprotein formulations cause macroscopically similar levels of irritationwhen compared to injections of equivalent volumes of saline solution.

The protein formulations can exhibit increased bioavailability comparedto the otherwise same protein formulation without the viscosity-reducingorganophosphate(s) when administered via subcutaneous or intramuscularinjection. “Bioavailability” refers to the extent and rate at which thebioactive species, e.g. a mAb reaches circulation or the site of action.The overall bioavailability can be increased for SC or IM injections ascompared to the otherwise same formulations without theviscosity-lowering organophosphate(s). “Percent bioavailability” refersto the fraction of the administered dose of the bioactive species whichenters circulation, as determined with respect to an intravenouslyadministered dose. One way of measuring the bioavailability is bycomparing the “area under the curve” (AUC) in a plot of the plasmaconcentration as a function of time. The AUC can be calculated, forexample, using the linear trapezoidal rule. “AUC_(0-t)”, as used herein,refers to the area under the plasma concentration curve from time zeroto a time, t, later. The time will typically be measured in days,although hours can also be used as will be apparent by context.“AUC_(∞)”, as used herein, refers to the area under the plasmaconcentration curve from time zero to a time where the plasmaconcentration returns to baseline levels. One way of measuring thebioavailability is by comparing the “area under the curve” (AUC) in aplot of the plasma concentration as a function of time. The AUC can becalculated, for example, using the linear trapezoidal rule. “AUC_(∞)”,as used herein, refers to the area under the plasma concentration curvefrom time zero to a time where the plasma concentration returns tobaseline levels. “AUC_(0-t)”, as used herein, refers to the area underthe plasma concentration curve from time zero to a time, t, later, forexample to the time of reaching baseline. The time will typically bemeasured in days, although hours can also be used as will be apparent bycontext. For example, the AUC can be increased by more than 10%, 20%,30%, 40%, or 50% as compared to the otherwise same formulation withoutthe viscosity-lowering organophosphate(s) and administered in the sameway.

As used herein, “t_(max)” refers to the time after administration atwhich the plasma concentration reaches a maximum.

As used herein, “C_(max)” refers to the maximum plasma concentrationafter dose administration, and before administration of a subsequentdose.

As used herein, “C_(min)” or “C_(trough)” refers to the minimum plasmaconcentration after dose administration, and before administration of asubsequent dose.

The C_(max) after SC or IM injection may be less, for example, at least10%, more preferably at least 20%, less than the C_(max) of anintravenously administered dose. This reduction in C_(max) may alsoresult in decreased toxicity.

The pharmacokinetic and pharmacodynamic parameters may be approximatedacross species using approaches that are known to the skilled artisan.

The pharmacokinetics and pharmacodynamics of antibody therapeutics candiffer markedly based upon the specific antibody. An approved murine mAbwas shown to have a half-life in humans of ˜1 day, while a human mAbwill typically have a half-life of 25 days (Waldmann et al., Int.Immunol., 2001, 13:1551-1559). The pharmacokinetics and pharmacodynamicsof antibody therapeutics can differ markedly based upon the route ofadministration. The time to reach maximal plasma concentration after IMor SC injection of IgG typically ranges from 2 to 8 days, althoughshorter or longer times may be encountered (Wang et al., Clin. Pharm.Ther., 2008, 84(5):548-558). The pharmacokinetics and pharmacodynamicsof antibody therapeutics can differ markedly based upon the formulation.Protein formulations containing organophosphates can exhibit improvedpharmacokinetics for SC or IM injections as compared to the otherwisesame formulations without the organophosphate(s). The C_(max) after SCor IM administration can be decreased as compared to the otherwise sameformulation without the viscosity-lowering organophosphate(s) andadministered intravenously. For example, C_(MAX) can be decreased bymore than 1 day, 2 days, 3 days, or 4 days or can be decreased by morethan 10%, 20%, 30%, 40%, or 50% compared to the C_(MAX) for theotherwise same formulation without the viscosity-loweringorganophosphates and administered intravenously. The overallbioavailability can be increased for SC or IM injections as compared tothe otherwise same formulations without the viscosity-loweringorganophosphate(s). The overall bioavailability can be assessed bycomparing one or more AUC values or by comparing the percentbioavailability computed, for instance, with respect to theintravenously administered dosage. For example, the AUC can be increasedby more than 10%, 20%, 30%, 40%, or 50% as compared to the otherwisesame formulation without the viscosity-lowering organophosphate(s) andadministered intravenously.

The low-viscosity protein formulations can allow for greater flexibilityin dosing and decreased dosing frequencies compared to those proteinformulations without the viscosity-lowering organophosphate(s). Forexample, by increasing the dosage administered per injection multiplefold, the dosing frequency can in some embodiments be decreased fromonce every 2 weeks to once every 6 weeks.

The protein formulations, including, but not limited to, reconstitutedformulations, can be administered using a heated and/or self-mixingsyringe or autoinjector. The protein formulations can also be pre-heatedin a separate warming unit prior to filling the syringe.

i. Heated Syringes

The heated syringe can be a standard syringe that is pre-heated using asyringe warmer. The syringe warmer will generally have one or moreopenings each capable of receiving a syringe containing the proteinformulation and a means for heating and maintaining the syringe at aspecific (typically above the ambient) temperature prior to use. Thiswill be referred to herein as a pre-heated syringe. Suitable heatedsyringe warmers include those available from Vista Dental Products andInter-Med. The warmers are capable of accommodating various sizedsyringes and heating, typically to within 1° C., to any temperature upto about 130° C. In some embodiments the syringe is pre-heated in aheating bath such as a water bather maintained at the desiredtemperature.

The heated syringe can be a self-heating syringe, i.e. capable ofheating and maintaining the liquid formulation inside the syringe at aspecific temperature. The self-heating syringe can also be a standardmedical syringe having attached thereto a heating device. Suitableheating devices capable of being attached to a syringe include syringeheaters or syringe heater tape available from Watlow ElectricManufacturing Co. of St. Louis, Mo., and syringe heater blocks, stageheaters, and in-line perfusion heaters available from Warner Instrumentsof Hamden, Conn., such as the SW-61 model syringe warmer. The heater maybe controlled through a central controller, e.g. the TC-324B or TC-344Bmodel heater controllers available from Warner Instruments.

The heated syringe maintains the liquid protein formulation at aspecified temperature or to within 1° C., within 2° C., or within 5° C.of a specified temperature. The heated syringe can maintain the proteinformulation at any temperature from room temperature up to about 80° C.,up to about 60° C., up to about 50° C., or up to about 45° C. as long asthe protein formulation is sufficiently stable at that temperature. Theheated syringe can maintain the protein formulation at a temperaturebetween 20° C. and 60° C., between 21° C. and 45° C., between 22° C. and40° C., or between 25° C. and 37° C. By maintaining the proteinformulations at an elevated temperature during injection, the viscosityof the liquid formulation is decreased, the solubility of the protein inthe formulation is increased, or both.

ii. Self-Mixing Syringes

The syringe can be self-mixing or can have a mixer attached. The mixercan be a static mixer or a dynamic mixer. Examples of static mixersinclude those disclosed in U.S. Pat. Nos. 5,819,988, 6,065,645,6,394,314, 6,564,972, and 6,698,622. Examples of some dynamic mixers caninclude those disclosed in U.S. Pat. Nos. 6,443,612 and 6,457,609, aswell as U.S. Patent Application Publication No. US 2002/0190082. Thesyringe can include multiple barrels for mixing the components of theliquid protein formulation. U.S. Pat. No. 5,819,998 describes syringeswith two barrels and a mixing tip for mixing two-component viscoussubstances.

iii. Autoinjectors and Pre-filled Syringes of Protein Formulations

The liquid protein formulation can be administered using prefilledsyringe autoinjector or a needleless injection device. Autoinjectorsinclude a handheld, often pen-like, cartridge holder for holdingreplaceable pre-filled cartridges and a spring based or analogousmechanism for subcutaneous or intramuscular injections of liquid drugdosages from a pre-filled cartridge. Autoinjectors are typicallydesigned for self-administration or administration by untrainedpersonnel. Autoinjectors are available to dispense either single dosagesor multiple dosages from a pre-filled cartridge. Autoinjectors enabledifferent user settings including inter alia injection depth, injectionspeed, and the like. Other injection systems can include those describedin U.S. Pat. No. 8,500,681.

The lyophilized protein formulation can be provided in pre-filled orunit-dose syringes. U.S. Pat. Nos. 3,682,174; 4,171,698; and 5,569,193describe sterile syringes containing two-chambers that can be pre-filledwith a dry formulation and a liquid that can be mixed immediately priorto injection. U.S. Pat. No. 5,779,668 describes a syringe system forlyophilization, reconstitution, and administration of a pharmaceuticalcomposition. In some embodiments the protein formulation is provided inlyophilized form in a pre-filled or unit-dose syringe, reconstituted inthe syringe prior to administration, and administered as a singlesubcutaneous or intramuscular injection. Autoinjectors for delivery ofunit-dose lyophilized drugs are described in WO 2012/010,832. Autoinjectors such as the Safe Click Lyo™ (marketed by Future InjectionTechnologies, Ltd., Oxford, U.K.) can be used to administer a unit-doseprotein formulation where the formulation is stored in lyophilized formand reconstituted just prior to administration. In some embodiments theprotein formulation is provided in unit-dose cartridges for lyophilizeddrugs (sometimes referred to as Vetter cartridges). Examples of suitablecartridges can include those described in U.S. Pat. Nos. 5,334,162 and5,454,786.

V. Methods of Purification and Concentration

The viscosity-lowering organophosphates can also be used to assist inprotein purification and concentration. The viscosity-loweringorganophosphate(s) and excipients are added to the protein in aneffective amount of viscosity-lowering organophosphate to reduce theviscosity of the protein solution. For example, the viscosity-loweringorganophosphate is added to a concentration of between about 0.01 M andabout 1 M, preferably between about 0.01 M and about 0.5 M, and mostpreferably between about 0.01 M and about 0.25 M.

The protein-organophosphate solution is then purified or concentratedusing a method selected from the group consisting ofultrafiltration/diafiltration, tangential flow filtration, centrifugalconcentration, and dialysis.

EXAMPLES

The foregoing will be further understood by the following non-limitingexamples.

All viscosities of well-mixed aqueous mAb solutions were measured usingeither a mVROC microfluidic viscometer (RheoSense) or a DV2T cone andplate viscometer (Brookfield; “C & P”) after a 5 minute equilibration at25° C. (unless otherwise indicated). The mVROC viscometer was equippedwith an “A” or “B” chip, each manufactured with a 50 micron channel.Typically, 0.10 mL of protein solution was back-loaded into a gastightmicrolab instrument syringe (Hamilton; 100 μL), affixed to the chip, andmeasured at multiple flow rates, approximately 20%, 40%, and 60% of themaximum pressure for each chip. For example a sample of approximately 50cP would be measured at around 10, 20, and 30 μL/min (approximately 180,350, and 530 s⁻¹, respectively, on an “A” chip) until viscositystabilized, typically after at least 30 seconds. An average absoluteviscosity and standard deviation was then calculated from at least thesethree measurements. The C & P viscometer was equipped with a CPE40 orCPE52 spindle (cone angle of 0.8° and 3.0°, respectively) and 0.50 mLsamples were measured at multiple shear rates between 2 and 400 s⁻¹.Specifically, samples were measured for 30 seconds each at 22.58, 24.38,26.25, 28.13, 30, 31.88, 45, 67.5, 90, 112.5, 135, 157.5, 180, 202.5,247, 270, 292.5, 315, 337.5, 360, 382, 400 s⁻¹, starting at a shear ratethat gave at least 10% torque, and continuing until instrument torquereached 100%. An extrapolated zero-shear viscosity was then determinedfrom a plot of dynamic viscosity versus shear rate for the samplesmeasured on a DV2T cone and plate viscometer. The extrapolatedzero-shear viscosities reported are the average and standard deviationof at least three measurements.

Example 1: Organophosphates Lower the Viscosity of Concentrated AqueousSolutions of Biosimilar AVASTIN®

A commercially-obtained biosimilar AVASTIN® (100-400 mg) containingpharmaceutical excipients (Polysorbate 20, phosphate and citratebuffers, mannitol, and NaCl) was purified. First, Polysorbate 20 wasremoved using DETERGENT-OUT® TWEEN Medi Columns (G-Biosciences). Next,the resulting solutions were extensively buffer-exchanged into 20 mMsodium phosphate buffer (PB; pH 7.0) for PB samples and 2 mM PB (pH 7.0)for viscosity-reducing organophosphate samples and concentrated to afinal volume of less than 10 mL on Jumbosep centrifugal concentrators(Pall Corp.). Samples buffer exchanged into 2 mM PB were firstaliquoted. Then, an appropriate amount of viscosity-reducingorganophosphate solution (pH 7.0) was added to each aliquot such thatupon reconstitution with water, the final excipient concentration was0.10-0.25 M. The protein solutions were then freeze-dried. The driedprotein cakes, containing protein and viscosity-reducing organophosphate(and a negligible amount of buffer salts) were reconstituted to a finalvolume of approximately 0.1 mL and viscosity-reducing organophosphateconcentration as previously described. For samples buffer exchanged into20 mM PB (PB control samples), the collected protein solution wasfreeze-dried. The dried protein cakes, containing protein and buffersalts were reconstituted to a final volume of approximately 0.10-0.50mL. These samples were reconstituted using additional PB (pH 7.0)sufficient to bring the final concentration of PB to 0.25 M. The finalconcentration of mAb in solution was determined by a Coomassie proteinquantification assay by comparing unknown concentrations of samples to astandard curve of biosimilar AVASTIN®. Viscosities reported weremeasured on a RheoSense mVROC microfluidic viscometer. Using the sameprotocol, formulations containing biosimilar ERBITUX®, TYSABRI®,HERCEPTIN®, and REMICADE® were also prepared.

The data in Table 1 demonstrate that the viscosity of aqueous solutionsof biosimilar AVASTIN® can be reduced by at least 2-fold in the presenceof 0.10-0.25 M viscosity-reducing organophosphate. Viscosities over 200cP in the phosphate buffer were reduced to under 50 cP in some cases bythe addition of 0.10-0.25 M viscosity-reducing organophosphates. TPPdemonstrates the greatest viscosity reducing ability (lowestviscosities) of the viscosity-reducing organophosphates examined here.

TABLE 1 Viscosities of aqueous solutions of biosimilar AVASTIN ® in thepresence of various organophosphates at 25° C. and pH 7. [Excipient][mAb] Viscosity Excipient (M) (mg/mL) (cP) PB 0.25 235 397 ± 2  PB 0.25220 213 ± 10 PB 0.25 200 96.8 ± 0.9 TPP 0.25 210 44.5 ± 0.9 TPP 0.10 21647.6 ± 2.7 TPP 0.10 201 34.6 ± 2.1 ATP 0.10 217 80.7 ± 7.1 ATP 0.10 20952.3 ± 2.3 ADP 0.10 215 76.6 ± 2.2 AMP 0.10 206 63.2 ± 1.6 cAMP-Tris0.25 216  85.7 ± 10.7 cAMP-Tris 0.25 209 62.1 ± 1.8 dATP-Tris 0.10 213206 ± 13 dATP-Tris 0.10 196  127 ± 0.2 GTP 0.10 205 150 ± 12 dTTP-Tris0.10 221 207 ± 6  dGTP-Tris 0.10 217 238 ± 31 dCTP-Tris 0.10 217 325 ±13 NADP 0.25 186 242 ± 16 NADP 0.10 204 165 ± 23 Pyridoxal Phosphate0.25 200 171 ± 11 Phosphoenol pyruvate 0.25 193 >>200 Phosphocreatine0.25 231 >>200 PB: Phosphate Buffer; TPP: Thiamine Pyrophosphate; ATP:Adenosine Triphosphate; ADP: Adenosine Diphosphate; AMP: AdenosineMonophosphate; cAMP: Cyclic Adenosine Monophosphate; dATP: DeoxyAdenosine Triphosphate; dTTP: Deoxy Thymidine Triphosphate; dGTP: DeoxyGuanosine Triphosphate; dCTP: Deoxy Cytidine Triphosphate; NADP:Nicotinamide Adenine Dinucleotide Phosphate. All phosphates are sodiumsalts unless otherwise stated.

Example 2. Viscosity Reduction of Aqueous Solutions of BiosimilarAVASTIN is Dependent Upon Organophosphate Concentration

Aqueous solutions of a commercially-obtained biosimilar AVASTIN® wereprepared as described in Example 1. The dried protein cakes werereconstituted in phosphate buffer or water to a final volume of about0.1 mL and a final viscosity-reducing organophosphate concentration of0.02 M-0.5 M. The final concentration of mAb in solution was determinedby a Coomassie protein quantification assay by comparing unknownconcentrations of samples to a standard curve of biosimilar AVASTIN®.Viscosities reported were measured on a RheoSense mVROC microfluidicviscometer.

The data in Table 2 demonstrate that the viscosity of aqueous solutionsof biosimilar AVASTIN® is initially reduced by the addition ofviscosity-reducing organophosphates. However, as the viscosity-reducingorganophosphate concentration increases beyond a certain value, theaddition of more viscosity-reducing organophosphate can becomecounterproductive (leads to increased viscosity). For the aqueoussolutions of biosimilar AVASTIN® examined here, the viscosity begins toincrease for concentrations above approximately 0.20 Mviscosity-reducing organophosphate.

TABLE 2 Viscosities of aqueous solutions of biosimilar AVASTIN ® in thepresence of varying concentrations of organophosphates [biosimilar[Excipient] AVASTIN ®] Viscosity Excipient (M) (mg/mL) (cP) PB 0.25 220213 ± 10 PB 0.25 180 ~60-70* TPP 0.10 176 21.1 ± 1.7 TPP 0.20 181 25.6 ±0.6 TPP 0.30 191 129 ± 5  TPP-APMI 0.02 213 69.5 ± 2.8 TPP-APMI 0.10 19123.2 ± 0.9 TPP-APMI 0.20 180 20.9 ± 0.4 TPP-APMI 0.30 182 33.3 ± 0.2TPP-APMI 0.50 180 111 ± 1  cAMP-Tris 0.10 171 22.0 ± 2.2 cAMP-Tris 0.40176 39.2 ± 4.3 cAMP-Tris 0.50 178 66.1 ± 0.4 *Interpolated value

Example 3. Organophosphates Lower the Viscosity of Many TherapeuticallyRelevant Monoclonal Antibodies

Aqueous solutions of a commercially-obtained biosimilar AVASTIN® wereprepared as described in Example 1. The dried protein cakes werereconstituted in phosphate buffer or water to a final volume of about0.10 mL and a final viscosity-reducing organophosphate concentration of0.02 M-0.50 M. The final concentration of mAb in solution was determinedby a Coomassie protein quantification assay by comparing unknownconcentrations of samples to a standard curve of biosimilar AVASTIN®.

Commercially-obtained TYSABRI® containing pharmaceutical excipients(sodium phosphate buffer, NaCl, Polysorbate 80) was purified, bufferexchanged, concentrated, dried, reconstituted, and analyzed in the samemanner. Commercially-obtained biosimilar ERBITUX® containingpharmaceutical excipients (sodium phosphate buffer, NaCl, Polysorbate80) was purified, buffer exchanged, concentrated, dried, reconstituted,and analyzed in the same manner. Commercially-obtained REMICADE®containing pharmaceutical excipients (sucrose, Polysorbate 80, sodiumphosphate buffer) was prepared as per instructions in the prescribinginformation sheet and purified, buffer exchanged, concentrated, dried,reconstituted, and analyzed in the same manner. Commercially-obtainedHERCEPTIN® containing pharmaceutical excipients (histidine buffer,trehalose, Polysorbate 20) was prepared as per instructions in theprescribing information sheet and purified, buffer exchanged,concentrated, dried, reconstituted, and analyzed in the same manner.Commercially-obtained biosimilar RITUXAN® containing pharmaceuticalexcipients (citrate buffer, sodium chloride, and TWEEN® 80) waspurified, buffer exchanged, concentrated, dried, reconstituted, andanalyzed in the same manner. Viscosities reported were measured on aRheoSense mVROC microfluidic viscometer.

The data in Table 3 demonstrate that viscosity-reducing organophosphatescan lower the viscosity of concentrated aqueous solutions of manytherapeutically relevant mAbs. cAMP-Tris reduces viscosity up to about9-fold in some cases.

TABLE 3 Viscosities of aqueous solutions of monoclonal antibodies in thepresence of organophosphates Organophosphate, 0.25M unless [mAb],Viscosity, mAb otherwise noted mg/mL cP Biosimilar PB 220 213 ± 10AVASTIN ® TPP (0.1M) 216 47.6 ± 2.7 cAMP-Tris 216  85.7 ± 10.7Riboflavin-5- 225 131 ± 4  phosphate (0.10M) Cidofovir 210 121 ± 2 hydrate (0.02M) REMICADE ® PB 213 1157 ± 22  PB 162 513 ± 15 TPP 2321773 ± 304 TPP (0.10M) 158 100 ± 9  TPP-APMI 223 316 ± 11 cAMP-Tris(0.10M) 156 59.7 ± 0.8 HERCEPTIN ® PB 239 122 ± 17 218 71.6 ± 3.9 TPP240 156 ± 3  TPP-APMI 221 60.5 ± 0.3 cAMP-Tris 235 79.5 ± 2.7 TYSABRI ®PB 237 182 ± 6  TPP 206 59.6 ± 1.6 TPP-APMI 238 76.3 ± 3.2 cAMP-Tris 22780.1 ± 2.4 Biosimilar PB 215 812 ± 49 ERBITUX ® TPP 215 55.6 ± 4.8Biosimilar Riboflavin-5- 237 492 ± 9  RITUXAN ® phosphate (0.10M) PB 199251 ± 1  TPP (0.10M) 193 102 ± 16 cAMP-Tris (0.10M) 199 62.6 ± 1.7

Unless expressly defined otherwise above, all technical and scientificterms used herein have the same meanings as commonly understood by oneof skill in the art. Those skilled in the art will recognize, or will beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

We claim:
 1. A liquid pharmaceutical formulation for injectioncomprising: (i) an antibody; (ii) a viscosity-reducing organophosphate,wherein the viscosity-reducing organophosphate comprises cyclicadenosine monophosphate (cAMP) or a pharmaceutically acceptable saltthereof; or adenosine triphosphate (ATP) or a pharmaceuticallyacceptable salt thereof; and (iii) a pharmaceutically acceptablesolvent; wherein the liquid pharmaceutical formulation, when in a volumesuitable for injection, has an absolute viscosity of from about 1 cP toabout 50 cP at 25° C. as measured using a cone and plate viscometer or amicrofluidic viscometer; and the absolute viscosity of the liquidpharmaceutical formulation is less than an absolute viscosity of acontrol composition comprising the antibody and the pharmaceuticallyacceptable solvent but without the viscosity-reducing organophosphate;and wherein the absolute viscosity is an extrapolated zero-shearviscosity.
 2. The liquid pharmaceutical formulation of claim 1, whereinthe viscosity-reducing organophosphate comprises cyclic adenosinemonophosphate (cAMP) or a pharmaceutically acceptable salt thereof. 3.The liquid pharmaceutical formulation of claim 1, wherein theviscosity-reducing organophosphate comprises adenosine triphosphate(ATP) or a pharmaceutically acceptable salt thereof.
 4. The liquidpharmaceutical formulation of claim 1, wherein the antibody has amolecular weight of from about 120 kDa to about 250 kDa.
 5. The liquidpharmaceutical formulation of claim 1, comprising from about 150 mg/mlto about 240 mg/ml of the antibody.
 6. The liquid pharmaceuticalformulation of claim 1, wherein the pharmaceutically acceptable solventis aqueous.
 7. The liquid pharmaceutical formulation of claim 1, furthercomprising one or more pharmaceutically acceptable excipients, the oneor more pharmaceutically acceptable excipients comprising a sugar, sugaralcohol, buffering agent, preservative, carrier, antioxidant, chelatingagent, natural polymer, synthetic polymer, cryoprotectant,lyoprotectant, surfactant, bulking agent, stabilizing agent, or anycombination thereof.
 8. The liquid pharmaceutical formulation of claim7, wherein the one or more pharmaceutically acceptable excipientscomprises a polysorbate, poloxamer 188, sodium lauryl sulfate, a polyol,a poly(ethylene glycol), glycerol, a propylene glycol, or a poly(vinylalcohol).
 9. The liquid pharmaceutical formulation of claim 7, whereinthe sugar alcohol is sorbitol or mannitol.
 10. The liquid pharmaceuticalformulation of claim 1 in a unit-dose vial, multi-dose vial, cartridge,or pre-filled syringe.
 11. The liquid pharmaceutical formulation ofclaim 1, wherein the liquid pharmaceutical formulation is isotonic tohuman blood serum.
 12. The liquid pharmaceutical formulation of claim 1,wherein the absolute viscosity is measured at a shear rate of at leastabout 0.5 s⁻¹ when measured using a cone and plate viscometer.
 13. Theliquid pharmaceutical formulation of claim 1, wherein the absoluteviscosity is measured at a shear rate of at least about 1.0 s⁻¹ whenmeasured using a microfluidic viscometer.
 14. The liquid pharmaceuticalformulation of claim 1, wherein the antibody is a monoclonal antibody.15. The liquid pharmaceutical formulation of claim 1, wherein the liquidpharmaceutical formulation is reconstituted from a lyophilizedcomposition.
 16. A method of administering a therapeutically effectiveamount of an antibody to a subject, the method comprising subcutaneouslyor intramuscularly injecting the liquid pharmaceutical formulation ofclaim 1 into the subject.
 17. The method of claim 16, wherein theinjecting produces a primary irritation index of less than 3 whenevaluated using a Draize scoring system.
 18. The method of claim 16,wherein the injecting has an injection force that is at least 10% lessthan an injection force for a control composition, the controlcomposition comprising the antibody and the pharmaceutically acceptablesolvent, but without the viscosity-reducing organophosphate, whenadministered in the same way as the liquid pharmaceutical formulation.19. The method of claim 16, wherein the injecting has an injection forcethat is at least 20% less than an injection force for a controlcomposition, the control composition comprising the antibody and thepharmaceutically acceptable solvent, but without the viscosity-reducingorganophosphate, when administered in the same way as the liquidpharmaceutical formulation.
 20. The method of claim 16, wherein theinjecting is performed with a needle between 27 and 31 gauge indiameter.
 21. The method of claim 20, wherein the injecting has aninjection force that is less than 30 N with a 27 gauge needle.
 22. Themethod of claim 16, wherein the injecting is performed with a heatedsyringe, a self-mixing syringe, an auto-injector, a pre-filled syringe,or combinations thereof.
 23. The method of claim 16, wherein the liquidpharmaceutical formulation has a volume equal to or less than about 1.5mL for the subcutaneous injecting, or equal to or less than about 3 mLfor the intramuscular injecting.
 24. A method of preparing the liquidpharmaceutical formulation of claim 1 comprising the step of combiningthe antibody, the pharmaceutically acceptable solvent, and theviscosity-reducing organophosphate.
 25. A lyophilized compositioncomprising: (i) an antibody; (ii) a viscosity-reducing organophosphate,wherein the viscosity-reducing organophosphate comprises cyclicadenosine monophosphate (cAMP) or a pharmaceutically acceptable saltthereof; or adenosine triphosphate (ATP) or a pharmaceuticallyacceptable salt thereof; and (iii) a pharmaceutically acceptableexcipient.
 26. The lyophilized composition of claim 25, wherein, oncereconstituted, the antibody has a concentration of at least 100 mg/ml.27. The lyophilized composition of claim 25, wherein theviscosity-reducing organophosphate comprises cyclic adenosinemonophosphate (cAMP) or a pharmaceutically acceptable salt thereof. 28.The lyophilized composition of claim 25, wherein the viscosity-reducingorganophosphate comprises adenosine triphosphate (ATP) or apharmaceutically acceptable salt thereof.